Fig. 2

Fig. 2 Morpholino knockdown of zebrafish Sema5A results in delay in CaP axon extension and axonal branching and is rescued by full-length rat Sema5A mRNA. (A–C) Lateral views of whole mount antibody labeling with znp1 of sema5A MO injected 26 hpf embryo (B, C) and uninjected WT sibling (A). Dashed white line indicate first intermediate target. Arrowheads indicate delayed axons. Arrow indicate an aberrant branch (D) RT-PCR showing efficacy of sema5A splice site MO in inducing aberrant splicing of sema5A mRNA in MO-injected embryos compared to uninjected wild type embryos. (E) Axon position was scored using different landmarks along the axon pathway in a dorsal to ventral direction. The landmarks on the x-axis are the horizontal myoseptum (HM), ventral edge of the notochord (VNC), proximal portion of the ventral muscle (PVM) and distal portion of the ventral muscle (DVM). (F) The axonal branching phenotype was scored by classifying branched axons into slight, moderate or severe branching. Data were quantified in sema5A MO injected embryos (n = 2020 axons, 101 embryos), sema5A MO and rat full-length sema5A RNA co-injected embryos (n = 2580, 129 embryos), WT uninjected (n = 1640 axons, 89 embryos) and control MO injected embryos (n = 2360 axons, 118 embryos) with embryos obtained from at least three separate experiments. Error bars represent confidence interval for proportions at 95% confidence. Asterisks indicate significant difference between 18 ng sema5A MO-injected embryos and sema5a MO + rat sema5a RNA injected embryos at p < 0.001 using one-way ANOVA.