The level of RTN3 expression plays a role in PGC migration to the gonadal region in zebrafish. (A) Specificity analysis of the PGCs labeled with EGFP. Embryos were collected at 30 h post-fertilization (hpf), and whole-mount fluorescence immunohistochemistry was performed using an anti-Vasa antibody to specifically label the germ cells; (B) PGC migration in wild-type embryos. Wild-type embryos were collected at about 24 hpf, oriented in agarose on a confocal dish, and screened using confocal microscopy; (C) Ectopic PGCs in RTN3 and CXCR4b mRNA injected embryos. Embryos were injected at the one-cell stage with 300 pg CXCR4b or 250 pg RTN3 synthetic mRNA per embryo. For controls, 1 nL of rhodamine was injected. After approximately 30 h, embryos were collected, and the positions of the PGCs were observed using confocal microscopy; (D) The percentages of embryos with mislocalized PGCs. Increasing the level of RTN3 or CXCR4b expression affected the number of ectopic PGCs compared with the control group; (E) Quantitative analysis of the numbers of PGCs in the experimental and control embryos. A total of 50–100 embryos were analyzed for each group. Error bars represent the mean ± SEM, * p < 0.05. Results are representative of three independent experiments. Scale bar, 20 μm for panel A and 100 μm for panels B, C1, C2, and C3. The arrows in B and C indicate PGCs.