Rottbauer 1
Movie Legends
Movie 1. dead beat renders the cardiac ventricle non-contractile.
After 72 hours of development, both cardiac chambers, the ventricle (V) and the atrium (A), contract vigorously in wild-type (wt) zebrafish embryos. In contrast, ded mutant embryos progressively lose ventricular contractility, and by 72 hours the ventricle becomes completely silent, while the atrium continues to contract normally. By that time the ventricular chamber tends to collapse and the atrium appears rather stretched due to increasing pericardial effusion.
Movie 2. dead beat acts cell-autonomously in the ventricular myocardium.
Mosaic hearts were generated by transplanting labelled (fluorescent) wild-type (wt) cells into deadbeat (ded) mutant embryos. Labelled wild-type cells (fluorescent) integrate normally in ded -/- ventricles and contract actively, even long after ded ventricular cardiomyocytes stop contracting.
Movie 3. Cardiac specific, mosaic over-expression of zebrafish PLC1 rescues the deadbeat ventricular contractility defect.
Wild-type zebrafish PLC1 expression was directed to myocardial cells of the heart using the cardiomyocyte-specific zebrafish myosin-light chain 2 (zMLC2) promoter. The expression construct was modified, such that the fluorescent marker nucDsRed2 is co-expressed with wild-type PLC1, and injected into ded -/- embryos, generating mosaic hearts. Cardiomyocytes, that express zebrafish PLC1 and nucDsRed2 under the control of zMLC2 contract vigorously in an otherwise silent ded ventricle.
Movie 4. VEGF and its receptor FLT-1 are essential for zebrafish cardiac ventricularcontractility.
VEGF or FLT-1 gene function was inhibited by injection of Morpholino-modified antisense oligonucleotides against VEGF (MO-vegf) or FLT-1 (MO-flt-1). After 72 hours of development, MO-vegf as well as MO-flt-1 injected embryos lose their cardiac ventricular contractility, whereas the atrium continues to contract. Thus, both MO-vegf as well as MO-flt-1 injection phenocopies the dead beat (ded) cardiac phenotype.
Movie 5. Blockage of VEGF receptors by PTK787/ZK222584 after 48 hpf of zebrafish development results in the loss of ventricular contractility of the heart.
VEGF receptor function was inhibited by bathing 48 hours old wild-type embryos with PTK787/ZK222584. By 48 hpf, both heart chambers were vigorously beating and blood cells were circulating through the arterial and venous vascular bed. However, as shown in the movie, 24 hours after incubation the bathed embryos loose their ventricular contractility, whereas the atrium continues to beat and blood cells are still circulating through the intact vascular bed.