| |
Figure 4. delta-Np63 is required for epidermal initial development. A: Both blastomeres of two-cell stage embryos were injected with delta-Np63(Ct)-GFP mRNA (3.5 ng/embryo). δNp63(Ct)-GFP was coinjected with CoMO (40 ng/embryo) or with δNp63MO (10, 30 or 40 ng/embryo). Injected and control embryos were cultured until stage 16 and processed for total protein extraction. delta-Np63(Ct)-GFP protein was detected by western blotting using an anti-green fluorescent protein (GFP) antibody. Each lane was loaded with 100 μg of total protein. delta-Np63MO inhibits translation of δNp63 in a dose dependent manner. B–G: delta-Np63MO (40 ng/embryo; B–F) or CoMO (40 ng/embryo; G) was injected into one blastomere of 8- to 16-cells stage embryos. Embryos were fixed at stage 16, and the expression of different genes was analyzed by in situ hybridization. B–D,G: Dorsal view, anterior at the top. F: Anterior view, dorsal at the top. B′,C′,E: Transverse sections. Arrowhead indicates the injected side evidenced by the linage tracer FLDx. Dashed lines indicate the midline. Brackets indicate the width of the neural plate. B,E,F: δNp63-depleted embryos fail to correctly express epidermal markers XK81a (B), Rexp52 (E), and cement gland marker XAG (F). C,D: Neural plate marker Sox2 and neural crest marker FoxD3 are expanded in the delta-Np63MO-injected side of embryos. F: Injection of control morpholino (CoMO) showed no effect on FoxD3 expression. np, neural plate.Download figure to PowerPoint |
