Has2 Expression in CPCs Is Required for Jogging and Rotation of the Cardiac Cone (A–L) One- and two-color ISH (probes indicated at bottom) on wild-type embryos (A–G, I, and K) or embryos injected with a southpaw MO (J) or a has2 MO (H and L) during various stages (indicated at the top) of heart tube formation. Evident is the dynamic expression pattern of has2 in the CPCs. All images are dorsal views with anterior to the top and left to the left. (M and N) Combined ISH with an antisense lefty2 riboprobe (blue) and anti-tropomyosin antibody staining (brown, labeling all cardiomyocytes) of a control MO-injected embryo (M) or has2 MO-injected embryo (N). (O) Selected images of a confocal time-lapse recording of a tg(cmlc2:gfp) embryo injected with a has2 MO starting at the 23-somite stage. Individual GFP-positive cells were tracked and color coded according to their location within the cardiac field at the 23-somite stage. Dorsal views with anterior to the top. (P) Displacement rates of CPCs localized at different positions within the cardiac field (color corresponds to position indicated in the confocal image) calculated from time-lapse recording shown in (O) (n = 55, mean ± SEM). Evident is the absence of leftward displacement of the CPCs and their equal displacement rates. (Q) Selected images of a confocal time-lapse recording shown in (O) with individual cells marked (red, light blue, dark blue, and green for atrial cells located lateral and pink for ventricular cells located medial) and tracked over a 250 min period. (R) Quantification of clockwise cardiac cone rotation in wild-type and has2 MO-injected embryos (mean ± SEM).
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