Figure 5

FO promotes vertebrae formation in zebrafish larvae. (A) Zebrafish larvae (3 dpf) were treated with FO (50 µg/mL and 100 µg/mL) and GP (4 mM) for 6 days (i.e., until 9 dpf) and then stained with calcein to visualize vertebrae formation. Relative intensity of calcein (bottom, left) and relative bone area (bottom, middle) were calculated using the Image J software and normalized against the GP-treated group. The number of vertebrate in each fish was manually counted. All of them are shown in the yellow box. Total fish length is 3.4 ± 0.4 mm. (B) Under the same experimental conditions, total mRNA from the 9 dpf zebrafish larvae was subjected to RT-PCR in order to determine zALP, zRUNX2a, zRUNX2b, zCol1a1, zOCN, zBMP2, and zBMP4 expression. zβ-Actin was used as a house keeping gene. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001, ** p < 0.01, and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas and GP; β-glycerophosphate.