Fig. 4

Fig. 4. wwtr1 is directly regulated by Foxc1a and can partially rescue zebrafish ventricular chamber maturation defects. A: A schematic diagram (top) of the zebrafish wwtr1 promoter. The dual-luciferase reporter assays (bottom) show that the 1550 bp, 935 bp, and 662 bp promoters of wwtr1 all responded to Foxc1a. B: The chromatin immunoprecipitation assays indicate that there are at least three binding sites for Foxc1a in the 1189 bp wwtr1 promoter. C: A dual-luciferase reporter assay performed with mutated wwtr1 promoters (P1, P2, P3, and P1-3 deletion). D: Fluorescence images of representative 96 hpf hearts from WT and foxc1a nju18 in the VD and VS phases after microinjection of wwtr1 mRNA. The dashed line represents the short axis of the ventricle. ‘-inj’ indicates embryos that were microinjected with wwtr1 mRNA at 1-cell stage. E: A scatter plot shows the shortening fraction in WT (n = 21), foxc1a nju18 (n = 25), WT-inj (n = 24), and foxc1a nju18-inj (n = 21) embryos. F: Maximum intensity projections of confocal z-stacks show representative 96 hpf hearts of WT-inj and foxc1a nju18-inj zebrafish. G: A scatter plot shows the number of ventricular cells in WT (n = 19), foxc1a nju18 (n = 10), WT-inj (n = 19), and foxc1a nju18-inj (n = 18) embryos. H: A scatter plot shows the pericardial edema indexes of WT (n = 20), foxc1a nju18 (n = 29), WT-inj (n = 24), and foxc1a nju18-inj (n = 30) embryos at 108 hpf. All images are ventral views with the head oriented upward. I: The diagram shows that the percentage of embryos with an abnormal trabecular phenotype was reduced after microinjection of wwtr1 mRNA. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001; ns, P > 0.05; Error bars represent the standard deviation. Scale bars, 50 μm. TSS, transcription start site; NC, negative control site; V, ventricle; A, atrium; VD, ventricular diastole; VS, ventricle systole.