Fig. 3

Expression of tyrosine hydroxylase (th) and fin movement frequency in ccdc141 and vstm2l morphants. In situ hybridization was conducted with tyrosine hydroxylase antisense RNA probe. At 3 dpf, (A) in ccdc141 5’UTR (MO1) morphants, the distribution of th is dispersed and the th-expressing amacrine cells (red arrows) are decreased; in vstm2l splicing (MO2) morphants, lower th expression in dorsal pretectum (red asterisk), DC7 neurons (red square) and amacrine cells was observed; and the distribution of th in sympathetic superior cervical ganglion (SCG, green rectangle) is decreased and dispersed. Of note, the th expression in locus coeruleus (LC) and medulla oblongata (MeO) neurons does not alter. For quantification before th in situ experiments, the morphants were separated into groups according to their phenotypic severity. The results showed that fewer th-expressing amacrine cells were observed in every group, including wt-like (P1) group in (B) 4.5 dpf ccdc141 MO1 morphants and (C) 4 dpf vstm2l MO2 morphants, whose statistical data are shown in (D) embryos injected with 1 ng ccdc141 MO1 morpholino and (E) embryos injected with 16 ng vstm2l MO2 morpholino, respectively. Note that (F) amacrine cell deceasing phenotype was rescued by co-injecting ccdc141 mRNA into ccdc141 morphants. G Hyperkinetic movements were observed in vstm2l MO2 morphants with significant differences. H The ccdc141 MO1 morphants had a trend of hyperkinetic movements, though the P value was not significant. (In these experiments, ccdc141 MO1 morphants were injected with 0.5 ng ccdc141 MO1 morpholino, and vstm2l MO2 morphants were injected with 16 ng vstm2l MO2 morpholino. Only wild-type like embryos were used to conduct experiments.) (N number for (A) AB = 2, ccdc141 MO1_1ng = 7, AB = 4, vstm2l MO2_16ng = 41, vstm2l MO2_16ng_ severe phenotype = 9. (D) AB = 5, MO-P1 = 6, MO-P2 = 24, MO-P3 = 2, MO = 32. (E) AB = 13, MO-P1 = 6, MO-P2 = 17, MO-P3 = 11, MO-P4 = 4, MO = 38. (F) AB = 21, MO1 = 18, MO1 + mRNA = 25. G vstm2l AB = 5, vstm2l MO2 = 4. H ccdc141 AB = 10, ccdc141 MO1 = 9.) (Mann–Whitney U test was used for comparisons of unpaired nonparametric variables. All calculated P-values were two-tailed, and statistical significance was defined as P-value less than 0.05. Symbol meaning: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****: p ≤ 0.0001)