miR499 deficiency delays IBs ossification and modulates muscle gene expression and locomotion. (A) qPCR analysis of sox6 and myosin-related genes in muscle tissues of WT and miR499−/− zebrafish at 30 dpf. (B,C) Tail-beat frequency and (C) stride length of WT and miR499−/− zebrafish at 30 dpf. Tf: Tail-beat frequency, Stride length was normalized by Body Length. (D) qPCR analysis of osteogenic genes in muscle tissues of WT and miR499−/− zebrafish at 30 dpf. (E) Alizarin red staining of IBs in WT and miR499−/− zebrafish at 30, 40, and 90 dpf. Scale bars = 1 mm. Arrows represent IBs. en: epineural bone; ep: epipleural bone. (F,G) Quantification of epineural bone (F) and epipleural bone (G) in WT and miR499−/− zebrafish across different body length ranges at 30 dpf. (H,I) Ossification rate of two IBs epineural bone and epipleural bone in miR499−/− mutant and WT. The relative length of IBs was calculated as: Relative length = Length of (epineural/epipleural bones)/Body length (BL). Growth rate curves of IBs were plotted based on these relative lengths across developmental stages. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. ns: no significance.
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