Fig 6

(A) HuC (elavl3) spatial expression in zebrafish at 48 hpf, determined by in situ hybridization. MO: MO injection; MO+ FL: MO and human FL TBC1D23 mRNA co-injection; MO+278/281/282: MO and human TBC1D23 L278A/Y281A/Y282A mutant mRNA co-injection; MO+236/237/239: MO and human TBC1D23 I236A/I237A/V239A mutant mRNA co-injection; MO+399/405: MO and human TBC1D23 C399S/R405A mutant mRNA co-injection. All injections are performed at the one-cell stage Top, lateral view; bottom, dorsal view. (B) The relative size of zebrafish midbrain. The size of the midbrain was measured from lateral view, and 5 to 10 embryos from each group were used for comparison. Data are presented as mean ± S.E.M. ****P < 0.0001. P values were calculated using one-way ANOVA, Tukey’s multiple-comparisons test. Experiments were triplicated, and the numerical data are included in S1 Data. (C) Relative transcription level of HuC at 48 hpf by semiquantitative RT-PCR analysis. Mean ± S.E.M. *P < 0.05. P values were calculated using one-way ANOVA, Tukey’s multiple-comparisons test. Experiments were triplicated, and the numerical data are included in S1 Data. (D) HuC (green) expression in Tg[HuC: GFP] transgenic zebrafish at 48 hpf, determined by immunofluorescence. Top, lateral view; bottom, dorsal view. (E) Morphology of CaP axons from embryos at 48 hpf that were injected MO and/or different mRNA. All injections are performed at the one-cell stage of the Tg[hb9: GFP]^ml2 transgenic zebrafish embryos. Arrows indicate abnormal branches. Lateral views and enlarged views are shown. (F) Statistical results of the branch number of CaP axons in embryos treated as in (E). For each group, approximately 45 axons from nine Tg[hb9: GFP]^ml2 transgenic zebrafish embryos are scored. Mean ± S.E.M. ****P < 0.0001,*P < 0.05. P values were calculated using one-way ANOVA, Tukey’s multiple-comparisons test. Experiments were triplicated, and the numerical data are included in S1 Data. FL, full-length; hpf, hours post fertilization; MO, morpholino oligonucleotide; ns, not significant; RT-PCR, reverse transcription PCR; WT, wild type.