Figure 3. Defective skeletal muscle development after knockdown of EBF2 and EBF3.

Two vegetal cells of eight-cell stage embryos were injected with control MO or EBF2 MO (2 MO) and EBF3 MO (3 MO), either alone or together. β-gal mRNA was coinjected as a marker of the injected side (light blue). At stage 39/40, myod expression was examined (A-H), and 12/101 antibody was used as a marker of skeletal muscle tissue (I-L). The left column (A, C, E, G, I, and K) shows the uninjected control side of the embryos. The right column (B, D, F, H, J, and L) shows the injected side, and (B and F) in some embryos there is more light blue staining in the pronephros, the functional larval kidney, which largely develops from the two vegetal cells that we targeted (Moody, 1987). All panels show lateral views. After injection of 2 MO or 3 MO, myod expression patterns show that the chevron shape of somites is abnormal (black arrows), the hypaxial muscle anlagen are smaller, and the migration distance is reduced (black arrowheads), compared to the uninjected side. The expression of myod in jaw muscle is also reduced (yellow arrows). When 2 MO and 3 MO were coinjected (H), these defects were more severe than 2 MO or 3 MO alone (D and F). Control MO has no effect (B). 12/101 antibody staining shows that when 2 MO and 3 MO were coinjected, somite segmentation is not complete, and the chevron shape of somites is abnormal (white arrows). Also jaw muscle differentiation is reduced (yellow arrow) and abdominal hypaxial muscle differentiation is strongly reduced (white arrowheads), while control MO shows a mild defect of only hypaxial muscle differentiation (J). To visualize the injected side after immunostaining, β-galactosidase antibody (not shown) was used for coimmunostaining along with 12/101 antibody.