Fig. 4

miR-143 regulates RA signaling. (A) An alignment of the sequences of miR-143 and its targets, raldh2 and rxrab. (B) Dual luciferase assay was performed with Cos7 cells cultured in the presence of 0.5% of serum. Expression of miR-143 repressed the reporters that contain the target motifs of raldh2 and rxrab (bars indicated by +), whereas the repressive effect of miR-143 was cancelled by mutating their sequences as shown in panel (A) (indicated by Raldh2 mut and RXRalpha mut). In the WT heart, rxrab (C) and raldh2 (D) were expressed in the atrial endocardium, not in the ventricular endocardium (blue arrowheads). In the morphants, both rxrab (C′) and raldh2 (D′) were ectopically induced in the ventricular endocardium (red arrowheads). When the beating of the WT hearts was stopped by BDM from 20 to 48 hpf, both rxrab (E) and raldh2 (E′) were induced in the ventricular compartment (red arrowheads), like the morphants. (F–K′) Raldh2 protein was detected by an antibody against it. In the WT, cytoplasmic staining was observed only in the atrial endocardial cells (F and G). Areas indicated by dotted lines were magnified in (H and H′). Raldh2 protein localizes in the cytoplasm of the atrial endocardium (H), not in the ventricular endocardium (H′). In the morphants, raldh2 was detected in both the atrial and ventricular endocardium (I and J). In magnified views, weak but distinct staining of raldh2 was observed even in the ventricular compartment (K′).