Heterozygous kcnj8+/V65M mutation induces CS-related cardiac anomalies and cerebral vasodilation in zebrafish. (A) Representative images illustrating the morphology of 5 dpf wild-type and kcnj8+/V65M mutants as seen from a left lateral (top) and dorsal (bottom) view. Boxes designate imaged areas that were used to assess cardiac function: the cardinal vein (1) and the heart (2). The ventricular area of the heart is highlighted, with the long axis and short axis of the ventricle indicated by dashed lines. a, atrium; ba, bulbous ateriosus; cv, cardinal vein; da, dorsal aorta; v, ventricle. (B) Quantification of cardiac function using individual characteristic confocal sections from a time series of the embryonic cardiac cycle at 5 dpf. Pericardial edema was quantified by measuring pericardial area using striking morphological landmarks, indicated by white boxes. Ventricular area was subtracted. Arrows show accumulation of fluid in kcnj8+/V65M mutants. Dotted red lines indicate ventricle (v) and bulbous arteriosus (ba). (C) Tracking of individual red blood cells (RBCs) measuring blood flow velocity in the cardinal vein. RBCs were tracked for ten frames using ImageJ (NIH) and the plugin MTrackJ (Meijering et al., 2012). One representative image of each genotype is shown. Black arrow indicates the direction of RBC movement. (D) Quantification of vascular dilations in a Tg(kdrl:GFP) background. Representative confocal images of the circular structure comprising the BCA and PCS in wild-type and heterozygous 5 dpf fish are outlined in red. The arrowheads indicate distinct regions of vasodilation. 3D reconstruction of vascular structure in Imaris was used to calculate vessel volume. (E) Ventricular area in heterozygous kcnj8+/V65M mutants. Representative heart histology of adult kcnj8+/V65M mutants and respective wild-type siblings after H&E staining. Exemplary depiction of one WT and one kcnj8+/V65M heart. For assessment of ventricular chamber size, tissue sections showing the largest ventricular area were selected and area was quantified using ImageJ (NIH). For all graphs, significance was determined by two-tailed unpaired Student's t-test or Mann–Whitney two-tailed U-test: *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001. The black horizontal bar indicates the mean value for each condition. Sample sizes: (B) kcnj8+/+, n=21; kcnj8+/V65M, n=14; (C) kcnj8+/+, n=10; kcnj8+/V65M, n=7; (D) kcnj8+/+, n=12; kcnj8+/V65M, n=20; (E) kcnj8+/+, n=6; kcnj8+/V65M, n=6. Scale bars: (A) 1 mm (top and middle) and 50 µm (bottom); (B) 50 µm; (C) 10 μm; (D) 50 µm; (E) 500 µm. All embryos analyzed originated from group matings of adult zebrafish.