Fig. 3

Fig. 3 Effects of RASSF4 on tail curling, pronephric filtration, pronephric cystogenesis, and light-dependent swimming defects in larval zebrafish with Pkd2 KD.
A Representative WB data showing the expression of Pkd2 and Rassf4 in 5-dpf zebrafish. B Effect of human RASSF OE on zebrafish tail curling induced by Pkd2 KD. Left panel, representative pictures presenting different severity levels of tail curling: normal (no curvature), moderate (0° < curvature angle < 90°), substantial (90°–180°), and severe (>180°). Right panel, distribution of the four tail curling severities under the indicated experimental conditions, with “Ctrl” indicating water injection. The total number of zebrafish used under each condition, from three independent experiments, is indicated. pkd2 KD: micro-injection of 200 pg pkd2-targeting sgRNA + 300 pg Cas9 mRNA; rassf4 OE: micro-injection of 100 pg rassf4 mRNA. ****p < 0.0001, and ns, not significant, by chi-square test. C Effect of Rassf4 KD on the severity distribution of tail curvature induced by Pkd2 KD. *p < 0.05, by chi-square test. D Effect of RASSF4 OE on the renal filtration defect induced by Pkd2 KD. Larval zebrafish at 3 dpf were injected with rhodamine dextran dye into the pericardial cavity. Representative fluorescence intensity measured at 3 and 24 h post-injection (hpi). Arrows indicate the heart. E Averaged residual fluorescence intensity at 24 hpi (relative to the value at 3 hpi). Data are presented as the mean ± SD, with n = 6–9. **p < 0.01, and ***p < 0.001, by unpaired Student’s t test. F Representative images of whole body and histological sections of 5-dpf larval zebrafish, showing the presence of cyst-like pronephric abnormalities, including tubule dilation and fluid around the tubule in fish with Pkd2 KD. G glomerulus, Pt pronephric tubule, Nc notochord. G Averaged cyst-like occurrence % obtained in 5-dpf zebrafish under indicated conditions, with cyst-like abnormalities identified as in (F), from three independent experiments. Cyst-like occurrence % was calculated from n = 20 to 28 fish for each condition in each experiment. Data are presented as the mean ± SD. **p < 0.01, by unpaired Student’s t test. H Representative swimming speed of 5-dpf larvae recorded at 30 s/sample under either the light (L) or dark (D) condition, each for 10 min. I Averaged swimming speed of 5-dpf larval zebrafish with or without Pkd2 KD and RASSF4 OE under the initial light for 90 min (left panel) and first dark (right panel) conditions. Data are presented as the mean ± SD, with n = 48 for each condition. ****p < 0.0001, by one-way ANOVA analysis.