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. 2011 Oct 15;138(20):4499–4509. doi: 10.1242/dev.067454

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© 2011.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which permits unrestricted non-commercial use, distribution and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the same Creative Commons License terms.

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Fig. 2. — Npnt knockdown disrupts heart development and is lethal. (A) Scheme of the effect of npnt splice-inhibitory morpholino MO2. Exons are represented by boxes and introns by lines. Dotted lines indicate the region targeted by MO1 or MO2 and arrows indicate RT-PCR primer positions. Exon E3 could not be detected. (B,C) RT-PCR analysis of mRNAs from control and MO2-injected zebrafish embryos at 52 hpf using npnt primers as indicated in A and gapdh primers (loading control) (n=3). MO2 inhibited npnt pre-mRNA splicing, resulting in intron insertion (B) and exon E5 deletion (C). (D) Western blot analysis of control and MO1- or MO2-injected embryos at 52 hpf (n=3). Both morpholinos efficiently knocked down the Npnt protein level. (E) Npnt knockdown resulted in 89±7.9% (mean ± s.e.m.) lethality at 7 dpf. (F) Lateral view of control, MO1- or MO2-injected embryos at 75 hpf. MO1 or MO2 injection resulted in pericardial edema (arrowheads). (G) Quantitative analysis. Valve formation and trabeculation are perturbed in 86±3.9% (mean ± s.e.m.) of morphant hearts at 110 hpf. (H) Confocal images of hearts from control and MO2-injected embryos from transgenic Tg(myl7:EGFP-HsHRAS)^s883 zebrafish at 80 hpf suggesting that the initiation of trabeculation is perturbed in npnt morphants. Red arrows indicate trabeculae. (I) Hematoxylin and Eosin (H&E)-stained sagittal sections of hearts from control and MO2-injected embryos at 110 hpf. In contrast to morphants, control embryos develop proper atrioventricular (AV) valve leaflets (white oval). Black arrowheads indicate the AV boundary; green arrows indicate expanded cardiac jelly. A, atrium; V, ventricle; PC, pericardium. Scale bars: 500 μm in F; 50 μm in H,I.