Advantages of use of zebrafish in cardiotoxicity research, which provide enormous information within a short time. (A) Dissecting microscope image of 3 hpf zebrafish embryos showing how easily accessible zebrafish embryos are to treat with chemicals at different developmental stages for desired periods; (B,C) Dissecting microscope images showing normal pericardium in the control embryos (yellow arrow) (B) and pericardial edema phenotype in 4 days post-fertilization (dpf) ethanol-treated zebrafish embryos (yellowstar) to help predict defective cardiogenesis (C); (D,E) Bright field images of Tg(myl7:GFP) embryos showing normal shaped two-chambered heart in control (D) and an almost linear heart in ethanol-exposed embryos (E), confirming heart malformation after ethanol exposure; (F,G) Confocal images of Tg(myl7:nlsKiKGR) embryos showing nuclei of cardiomyocytes in closely apposed bean-shaped atrium and ventricle in control embryos (F), fewer cardiomyocytes are seen in misshapen chambers of ethanol-treated embryos (G); and (H,I) Confocal images of Tg(fli1:EGFP) embryos show endocardial cells in normal endocardium in control embryos (H), fewer endocardial cells are seen in misshapen endocardium of ethanol-treated embryo (I).

Zebrafish studies discovered altered valve regulatory pathways due to embryonic ethanol exposure leading to persistent atrioventricular valve defects. (A) Schematic representation of atrio-ventricular canal (AVC) showing myocardium and endocardium layers. Bmp, Notch and Wnt signaling play critical roles during AVC differentiation. Ethanol exposure reduced Notch and Wnt activity at the AVC (represented by small dark brown arrows) during atrioventricular valve formation; (B) Schematic representation of the ventricle showing myocardium and endocardium layers. Ethanol exposure (3–24 hpf) increased Notch activity in the ventricle (represented by big dark brown arrows) during atrioventricular valve formation. Green arrow: normal condition; dark brown arrow: ethanol-exposed condition; (C) Schematic representation of atrium, ventricle and AVC (black arrow) of the control zebrafish heart at 50 hpf (during atrioventricular valve formation). Differentiated valve-forming cells (red) are localized at the AVC. Gray line represents myocardium layer; greenish-yellow line represents endocardial layer; (D) Schematic representation of the atrium, the ventricle and the AVC of the ethanol-treated (3–24 hpf) zebrafish heart at 50 hpf (during atrioventricular valve formation). Note that the shape of the heart is different from control. Differentiated valve-forming cells (pinkish-red), which do not exhibit all characteristics of normal valve cells are not restricted at the AVC. Those cells extend into the ventricle. The distance between myocardium and endocardium (the space containing cardiac jelly; black line) is more in ethanol-treated embryos. Gray line represents myocardium layer; greenish-yellow line represents endocardial layer; and (E,F) Wheat germ agglutinin-stained atrioventricular valves of two-month-old zebrafish shows four well-organized valve cusps in control fish (E), and small, deformed valve cusps in fish treated with ethanol during embryonic development (3–24 hpf).