Figure 4

Fluoride affects the features of clcn3 morphants. The zebrafish embryos were injected with clcn3 morpholinos or control morpholinos and then incubated in 38 ppm or 76 ppm NaF for five days. (A) Ratios of abnormal phenotypes of clcn3 morphants w/o fluoride treatment. The calculated number of zebrafish embryos was recorded in each column. (B) Craniofacial features of 5 dpf clcn3 morphants using Alcian blue–Alizarin red double staining. The head skeleton of 5 dpf control zebrafish larvae presents calcified pharyngeal teeth, whereas Meckel’s cartilage and the ceratohyal remain cartilaginous. In clcn3 morphants, the ceratohyal is malformed and the ceratohyal angle is altered. (C) Schematic map of zebrafish head for calculating. Ch, ceratohyal; 5th Cb, 5th ceratobranchial arches; M, Meckel’s cartilage; Pq, palatoquadrate; a, head length; b, head width; c, distance from Meckel’s cartilage to ceratohyal. (D) Comparison of skeleton and craniofacial parameters such as body length, head length, head width, the length of ceratohyal, Meckel’s cartilage and palatoquadrate. (E) Teeth characteristics of 5 dpf zebrafish larvae stained with Alcian blue–Alizarin red. The number and shape of teeth were altered in both clcn3 morphants and fluoride-treated zebrafish embryos. Alizarin red staining intensity was markedly reduced in these groups, with the greatest decrease observed in 76 ppm NaF-treated clcn3 morphants. (F) Ultrastructure characteristics of 5 dpf zebrafish teeth using SEM observation. clcn3 morphants and fluoride-treated zebrafish embryos exhibited a rough surface on the teeth. Fluoride treatment further worsened the tooth deformities in clcn3 morphants. White arrows point to the rough surface on enameloid surface. Scale bars represent 5 μm (upper panel) and 1 μm (lower panel), respectively. *, p < 0.05; **, p < 0.01; ***, p < 0.001.