Craniofacial and eye features in 7 dpf Alizarin Red S and Alcian Blue-stained sgms2a kd, sgms2b kd, and sgms2a+b kd larvae. (A) Craniofacial deformity. Craniofacial phenotype was classified into three categories: normal, mild deformity, and severe deformity. Representative images of each category are presented on the left side of the graph (left row: lateral view, right row: ventral view). Cartilage elements are stained in Alcian Blue and mineralized skeletal elements in Alizarin Red S. Data are shown as a bar chart. The p-values were generated by Fisher’s exact test and Bonferroni correction (sham larvae vs WT, sgms2a kd, sgms2b kd, and sgms2a+b kd larvae). (B) Representative ventral view image of Alizarin Red S and Alcian Blue-stained zebrafish larvae head region with measured parameters indicated. Measurements of parameters are presented in box plots (C-J). (C) Head length. (D) M angle. (E) Ch angle. (F) Pq-m angle. (G) Pq-ch angle. (H) Eye width. (I) IOD. (J) PS area. In (C-J), the p-values were generated by ordinary one-way ANOVA for multiple comparisons (sham larvae vs WT, sgms2a kd, sgms2b kd, and sgms2a+b kd larvae). (K) Eye deformity. Data are shown as a bar chart. Eye phenotype was classified into four categories: normal, cyclopia, hypotelorism, and uneven size. The number of zebrafish larvae with the phenotype are indicated in the x-axis. Representative images of each category are presented on the left side of the bar chart. ch, ceratohyal; iod, interocular distance; kd, knockdown; m, Meckel’s cartilage; pq, palatoquadrate; ps, parasphenoid bone. Scale bar = 100 μm (A and B), and 200 μm (K). p < .05 was considered statistically significant. Only significant p-values were presented.
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