(A) Tg(flk1:EGFP) zebrafish embryos injected with morpholinos (MOs) for novel candidate genes for CAD showed morphological heart abnormalities, such as peripheral edema at 3 days post-fertilization (arrows in the left panel, scale bar = 500 μm). Zebrafish embryos normally have hearts with a left ventricle (V) and right atrium (A), whereas the embryos injected with MOs related to CAD genes exhibited either no asymmetry or reversed V and A orientation (middle panels, scale bar = 200 μm). These embryos also exhibited malformed blood vessels in the trunk (asterisks in the right panel, scale bar = 200 μm). (B) MO-injected Tg(flk1:EGFP) zebrafish embryos were counted to quantify those that exhibited heart asymmetry. (C) MO-injected Tg(flk1:EGFP) zebrafish embryos were counted to quantify those that exhibited vascular defects. Over 20 MO-injected embryos per gene were counted for each analysis (A-C).

In vivo validation of candidate coronary artery disease (CAD) genes.

(a) The sequence of translation-blocking MOs targeting candidate CAD genes used for this study. (b) Tg(flk1:EGFP) zebrafish embryos were injected with morpholinos (MOs) for candidate CAD genes and compared with control MO-injected embryos (morphants). The majority of morphants, except for apod morphants, exhibit gross morphological abnormalities, including a small brain, heart edema, and curved tail, at 3 days post-fertilization (scale bar = 500 μm). (c) Diagrams show the representative heart defects, such as no asymmetry (midline) and reversed asymmetry between ventricle and atrium, at 3 days post-fertilization.