Figure 3.

Rad21 depletion causes cardiac valve defects and decreased ejection of blood from the heart. Zebrafish embryos were injected at the one-cell stage with 1 nl Phenol Red dye, or with 0.035 pmol Rad21 ATG MO, then raised as for Figure 2. Scale bars are 20 µm. (A and B) Time series of heart valves at 5 dpf, visualized using a transgenic Tg(5xERE:EGFP²⁶²) zebrafish line in which the heart valves are GFP-labeled. (A) Normal valve morphology and function in control embryos. The opening of the VB valve is oriented at right angles to the AV valve (white arrows). The AV and VB valves open and close alternately. Valve defects were never observed in controls (n = 10). (B) Example of the extreme heart valve defects observed in Rad21-depleted embryos. In three out of eight Rad21-depleted embryos analyzed, the VB valve failed to develop and the AV valve failed to close properly. In the remaining five out of eight Rad21-depleted embryos, both AV and VB valves developed, but were notably smaller compared with control valves, as shown in (D). In all Rad21-depleted embryos, the orientation of the VB valve was in line with the AV valve (white arrows), in contrast to the perpendicular orientation of the valves seen in controls. (C and D) Time series of blood pumping through the heart at 5 dpf, visualized using a double-transgenic Tg(gata1:dsred), Tg(5xERE:EGFP²⁶²) zebrafish line, in which erythrocytes are labeled red and the heart valves are green. (C) Normal heart function in a control embryo. Blood flows from the atrium (1) into the ventricle (2) in a contralateral direction, and from the ventricle intro the bulbus arteriosus (3) in a cranial direction. (D) Rad21-depleted embryo. The volume of blood pumped into both the ventricle and the atrium is reduced in Rad21-depleted embryos (see also Supplementary Material, Video S3). (E) Pictorial representation of the heart morphologies shown in C and D. AV, atrioventricular valve; VB, ventriculobulbar valve. 1, atrium; 2, ventricle; 3, bulbus arteriosus.