Notochord features in 7 dpf Alizarin Red S and Alcian Blue-stained sgms2a kd, sgms2b kd, and sgms2a+b kd larvae. (A) Representative lateral view whole-body images of Alizarin Red S and Alcian Blue-stained 7 dpf WT, sham, sgms2a kd, sgms2b kd, and sgms2a+b kd zebrafish larvae. Measurement of standard length is illustrated in the WT zebrafish. (B) Standard length. Data are shown as a box plot. 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). (C) Notochord phenotype. 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). The notochord phenotypes were classified into eight categories: normal, abnormal thickness, curved, local deformity/kink, abnormal thickness + curved, abnormal thickness + local deformity/kink, curved + local deformity, and kinks + bending. Representative images of the following notochord features: normal, abnormal thickness + local deformity/kink, curved + local deformity/kink, and kinks and bending, are presented on the left side of the bar chart. (D) Bending angle. Data are shown as a scatter dot plot with mean ± SD. The bending angles between the sgms2a kd, the sgms2b kd, and the sgms2a+b kd larvae were compared by ordinary one-way ANOVA for multiple comparisons. (E) Early vertebral element (notochord sheath) mineralization. Mineralization was classified into three categories: normal (at least 3 mineralized elements), decreased (less than 3 mineralized elements), and no mineralization (0 mineralized elements). The representative images of the categories are presented on the left side of the bar chart. 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). Abbreviation: kd, knockdown. Scale bar = 500 μm (A) and 100 μm (C-E). p < .05 was considered statistically significant. Only significant p-values were presented.
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