Heterozygote knockout of miRNA-9 did not affect M-cell axon regeneration and growth and development in zebrafish larvae. a Generation of miRNA-9± and Tg (Tol-056); miRNA-9+/− zebrafish. miRNA-9−/− can cause embryonic lethality. b, c Heterozygote knockout of miRNA-9 did not affect M-cell axon regeneration (control: 383.4 ± 22.37 μm, n = 17 fish; miRNA-9+/−: 390.1 ± 20.69 μm, n = 24 fish). White asterisk: ablation point; arrowhead, axon regeneration terminal. scale bar, 50 μm. p = 0.8283, assessed by unpaired t-test, ns, not significant. d, e Total lengths of M-cell axons from the cloaca to the end were not notably different among WT and heterozygous knockout larvae (control: 1464 ± 17.8 μm, n = 8 fish; miRNA-9+/−: 1491 ± 26.07 μm, n = 8 fish). White asterisk: ablation point; arrowhead, axon regeneration terminal. scale bar, 50 μm. p = 0.4173, assessed by unpaired t-test, ns, not significant. f, g Trajectory diagrams and motion distance statistics of free swimming within 1 h of miRNA-9+/− (control: 584.6 ± 34.03 cm, n = 24 fish; miRNA-9+/−: 545.3 ± 30.90 cm, n = 24 fish). p = 0.3973. Assessed by unpaired t-test. h, i Representative images of larvae from the wildtype and the miRNA-9+/− at 6 dpf (scale bar, 500 μm), and measured total body length from 4 to 6 dpf (4 dpf: control: 3.68 ± 0.0226 mm; miRNA-9+/−: 3.730 ± 0.0217 mm, p = 0.1189, n = 15 fish; 5 dpf: control: 3.993 ± 0.0252 mm; miRNA-9+/−: 3.984 ± 0.0326 mm, p = 0.8324, n = 15 fish; 6 dpf: control: 4.082 ± 0.0192 mm; miRNA-9+/−: 4.098 ± 0.0293 mm, p = 0.6459, n = 15 fish). Assessed by two-way ANOVA. ns, not significant
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