Retinoic Acid Signaling Is Essential for Valvulogenesis by Affecting Endocardial Cushions Formation in Zebrafish Embryos

Retinoic acid (RA) plays important roles in many stages of heart morphogenesis. Zebrafish embryos treated with exogenous RA display defective atrio-ventricular canal (AVC) specification. However, whether endogenous RA signaling takes part in cardiac valve formation remains unknown. Herein, we investigated the role of RA signaling in cardiac valve development by knocking down aldh1a2, the gene encoding an enzyme that is mainly responsible for RA synthesis during early development, in zebrafish embryos. The results showed that partially knocking down aldh1a2 caused defective formation of primitive cardiac valve leaflets at 108 hpf (hour post-fertilization). Inhibiting endogenous RA signaling by 4-diethylaminobenzal-dehyde revealed that 16-26 hpf was a key time window when RA signaling affects the valvulogenesis. The aldh1a2 morphants had defective formation of endocardial cushion (EC) at 76 hpf though they had almost normal hemodynamics and cardiac chamber specification at early development. Examining the expression patterns of AVC marker genes including bmp4, bmp2b, nppa, notch1b, and has2, we found the morphants displayed abnormal development of endocardial AVC but almost normal development of myocardial AVC at 50 hpf. Being consistent with the reduced expression of notch1b in endocardial AVC, the VE-cadherin gene cdh5, the downstream gene of Notch signaling, was ectopically expressed in AVC of aldh1a2 morphants at 50 hpf, and overexpression of cdh5 greatly affected the formation of EC in the embryos at 76 hpf. Taken together, our results suggest that RA signaling plays essential roles in zebrafish cardiac valvulogenesis.