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Morpholine antisense oligonucleotide treatment decreases expression of the flow-responsive gene klf2a results in valve dysgenesis. (A-D) Valve leaflets scored at 96 hpf show effects of klf2a MO. (A and C) Sham-injected embryos form normal heart valves. (B and D) klf2a MO-treated embryos display valve dysgenesis, often with a complete absence of valve leaflets. (C and D) Detailed views of valve morphology. (C) Control embryo has clearly distinguishable valve leaflets (arrows). (D) klf2a MO-treated embryo has no valve leaflets forming from the endocardium (arrow) (52%, n = 46). The proportions were significantly different at a level of significance ± = 0.01. Scale bars indicate 50 μm. (E and F) Average flow pattern at 48 hpf in controls (E) and klf2a morphants (F) showing that the RFF is unaffected in the mutants but that the heart rate is slightly decreased. (G-L) Expression of three marker genes at 48 hpf in normal and klf2a morphants. (G-J) nppa expression is normal in the klf2a morphants, showing that chamber specification occurs independently of klf2a. (I and J) bmp4 mRNA distribution at 48 hpf showing that expression is decreased in the MO-treated embryo in the AV node region at 48 hpf (n = 23, 40%; compare expression at arrow in panels [I and J]). (K and L) notch1b expression at 48 hpf decreases in the AV boundary of the klf2a morphants (n = 45, 71%). Arrows point to the AV boundary in all panels (G–L). (M) Summary of quantitative RT-PCR showing the expression level of flow-responsive genes in klf2a morphants. Expression of all genes decreases significantly, confirming the down-regulation of bmp4 and notch1b observed by ISH. **p<0.01, ANOVA. (N) Summary diagram of klf2a function during heart valve formation. klf2a acts as a transcriptional relay between the reversing flow generated by the circulating blood cells at the AV canal and several genes activated in the AV endothelial cells (such as notch1b, neuregulin1, and endothelin1). klf2a also affects the expression of bmp4, revealing a possible interaction between myocardium and endothelium essential for valve morphogenesis.
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