wnt10a is expressed during zebrafish embryonic development. (A) wnt10a mRNA expression was detected by qPCR analysis in zebrafish craniofacies at critical time points for tooth development (18hpf, 24hpf, 2dpf, 3dpf, 4dpf, and 5dpf). (B) Schematic of zebrafish structure and whole‐mount in situ hybridization of zebrafish embryos at 56hpf showing widespread and uniform expression of wnt10a mRNA.

Knockdown of wnt10a expression impairs tooth development at 5dpf and recapitulates human tooth agenesis phenotype. (A) Morpholino knockdown of zebrafish wnt10a. Target sites for translation‐blocking (TBMO, red triangle) and splice site‐blocking (SBMO, blue triangle) morpholinos. (B) Morpholinos were used to block the translation initiation complex (TB) or the locus involved in splicing pre‐mRNA (SB), or used as mismatch control (MM). Wnt10a protein production is disrupted at dotted frames for TB (red frame) and SB (blue frame). (C) Western blot analysis confirming knockdown efficiency. Wnt10a level was largely reduced in the MO‐injected zebrafish embryos compared to uninjected control (UIC) embryos. Beta‐actin was used as loading control. (D) UIC, wnt10a‐MO‐injected fish and mismatch‐MO‐injected embryos at 24hpf do not present gross body abnormalities. (E–H) Alcian blue and alizarin red staining of: (E) UIC, wnt10a morpholino‐injected (MO) zebrafish at 5dpf showing similar body structure and appearance, and (F,G) the presence of pharyngeal teeth in UIC (red arrow, left panel), but not in wnt10a‐MO‐injected embryos (right panel). (G) Higher magnification of dotted box in (F). (H) UIC control zebrafish embryos had no noticeable cartilage defects, whereas wnt10a‐MO‐injected fish presented with some cartilage abnormalities. (I, J) Mismatch morpholino‐injected embryos at 5dpf develop normally and present with pharyngeal teeth (red arrows) similarly to UIC embryos (embryos were cleared to enhance mineralized structures). Scale bar represents 150 µm.

Perturbation of wnt10a expression results in altered expression of additional tooth development genes at tooth bud formation stage (2 dpf). (A) Quantitative RT‐PCR analyses of msx1, dlx2b, eda, and axin2 genes in WT, mismatch‐MO injected (MM), wnt10a‐MO injected (MO), wnt10a RNA injected (wnt10a), and wnt10a mutant RNA with T328I and G248S mutations (corresponding to T357I and G213S in humans). No differences were observed in the expression of those genes between WT and MM‐injected embryos, whereas knockdown of wnt10a led to significantly decreased expression of dlx2b, eda, and axin2 (P = 0.001). In contrast, overexpression of wnt10a resulted in increased expression of all four genes (P < 0.02), although with significant differences only for msx1 (P = 0.01) and axin2 (P = 0.02). Injection of mutant wnt10a RNAs also perturbed expression of msx1, dlx2b, eda, and axin2, particularly the G248S mutant (0.002 < P < 0.02). Target gene expression was normalized to the expression of beta‐actin used as endogenous control. Fold changes were calculated in relation to controls by the 2^−∆∆Ct method and analyzed using Student's t test. P‐values ≤0.05 were considered statistically significant. (B,C) Overexpression of Wnt10a leads to a small/no eyes phenotype in wnt10a RNA‐injected fish (C) in comparison to WT (B) at 2 dpf. Embryos not injected with any RNA (D), injected with 1 pg zebrafish wnt10a RNA (E), 1 pg wnt10a RNA with T382I mutation (F), and 1 pg wnt10a RNA with G248S mutation (G).

Analysis of Zebrafish wnt10a Morphant Embryos.

wnt10a MO-injected fish showed similar body structure and appearance compared to uninjected fish. Developmental periods were classified based on the landmarks: segmentation period (10-24 h), pharyngula period (24-48 h), hatching period (48-72 h), and larval (3-29 days).

wnt10a Knockdown Causes Cartilage Abnormalities in Zebrafish.

Alcian blue and alizarin red staining of (A) Un-injected and (B) wnt10a MO-injected zebrafish at 5dpf. While un-injected fish have no noticeable cartilage defects, MO-injected fish present with a range of mild (top) to more severe (bottom) cartilage abnormalities.