Has2 knockdown rescues the endocardial phenotype in npnt morphants. (A) Confocal sections and schematics of whole-mount Alcam-stained (red, indicating myocardium and differentiated endocardial cells) MO2-injected, has2 MO + MO2-injected, control-injected and has2 MO-injected embryos from transgenic Tg(kdrl:EGFP)s843 zebrafish at 52 hpf. Red line, myocardium; green, endocardial cells; black dots, Alcam-positive endocardial cells. Npnt depletion caused an increase in differentiated AV endocardial cells (cuboidal in shape, Alcam positive and EGFP positive) (number of tiers e8, SAV; number of tiers e5, IAV). Knockdown of Has2 in npnt morphants or wild-type embryos reduced the number of tiers to two or fewer in SAV or IAV. Arrowheads indicate the AV boundary. (B) Quantitative analysis. Has2 knockdown in npnt morphants rescued the AV canal endocardium extension (mean ± s.e.m.). SAV, superior AV; IAV, inferior AV. (C) Bright-field images of 52 hpf MO2-injected and has2 MO + MO2-injected embryos after whole-mount in situ hybridization for notch1b, cspg2 and bmp4 expression (brackets). Note that Has2 knockdown reduced the expanded expression of notch1b but not of cspg2 or bmp4 in npnt morphants (arrows). A, atrium; V, ventricle. Scale bars: 50 μm.
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