Characterization of NXT2 mutant phenotypes. At 3 dpf, NXT2 mutants show pericardial edema (compare B and G to wild-type embryos A and D). By 4 dpf, both atrium and ventricle of NXT2 mutants show significant dilation (compare H to wild-type embryo E) with pericardial sac enlargement (C and H). At 5 dpf, NXT2 mutant heart appears to be lacking atrioventricular boundary (F, arrow) and shows blood accumulation in both cardiac chambers in enlarged heart sac (F) and /or peripheral circulation (I, tail vessel) whereas the wild type heart with a normal atrioventricular boundary (arrow) resides in a narrowed cardiac sac (inset in F). Sections of wild-type (J) and NXT2 mutant (K) hearts at 4 dpf show that ventricular wall of NXT2 mutant embryos is thinner and a space separating myocardium and endocardium in atrium is visible. In addition, we often observed mutant embryos lacking valve structure (arrows). RNA whole mount in situ hybridization analyses of myocardial differentiation in wild-type (L, M, N, R, T) and NXT2 mutant (O, P, Q, U, W) at 3 dpf. All ventral view, anterior to the top except S and V, which are lateral view, anterior to the left. Solid red line marks the site of the boundary between the ventricle (v) and the atrium (a). NXT2 mutants show normal expression of cardiac chamber-specific markers amhc (L and O), vmhc (M and P), cmlc2 (N and Q). At 3 dpf, versican (R and U) and bmp4 (T and W) are restricted to the atrioventricular boundary in wild-type embryos (black arrows), but, are diffusely expressed in both cardiac chambers in NXT2 mutants. Versican expression pattern in otoliths is not changed in mutant embryos (S and V, red arrows: heart). vv: veinous vessels; a: atrium; v: ventricle; av: atrioventricular boundary; ot: otoliths. wt: wild type; mutant: zNXT2 homozygous mutant embryos; dpf: days post fertilization.
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