The failure of klf2a expression in the AVC is blood flow-independent, and klf2a is aberrantly activated by Mef2c in the endocardium, which ultimately impedes valvulogenesis. (A–C)Vgll4b–/– embryos were treated with a high dose of gata1 MO, but no rescue effect was observed. (D,E)Trpv4 mRNA was injected alone or in combined with gata1 MO into vgll4b–/– embryos. (F,G)Klf2a MO and flk1:klf2a DN rescue assays in vgll4b–/– embryos. (H) The hearts were harvested respectively from 35 wild type and 35 vgll4b–/– embryos at 52∼54 hfp. Q-PCR analysis revealed that the expression level of klf2a and notch1b in vgll4b–/– hearts was elevated (0.34-fold and 3.2-fold inductions compared to controls). Error bars represent ± SD of at least three replicates. p values are denoted by asterisks; ∗P < 0.1, ∗∗∗P < 0.001 (Student’s t test). (I) Dual luciferase vectors each with a fragment of the zebrafish klf2a promoter (–1.5 kb) were co-transfected into HEK293T cells with empty vector pCS2+, a mef2cb expressing vector, mef2cb combined with wild type vgll4b or vgll4b ΔTDU2 expressing vector. Luciferase activity was detected and normalized to empty vector pCS2+ which was set to 1.0. Error bars represent ± SD of at least three replicates. p values are denoted by asterisks; ∗∗∗P < 0.001 (ANOVA test). (J) Schematic depiction of the aberrant Vgll4b-Mef2c regulation in valvulogenesis in vgll4b–/– zebrafish.
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