Figure 5 cont.

BH3-only proapoptotic gene Bad can regulate brain development between 24 and 48 hpf. (A) Dosage dependence of Bad knockdown (lanes 3–5 for 0.1, 0.2, and 0.4 mM, respectively) shown by Western blot analysis and compared with the uninjected group (lane 1) and the normal control Bad-M5 group (lane 2). The positive control MCF7 cell lysate is shown in lane 6. (B–D) Identification of morphological phenotype defects in the brain during Bad knockdown with dosage response. Each one-cell-stage embryo was injected with either 0.4 mM of control-MO or 0.1, 0.2, or 0.4 mM of Bad-MO. At 24 and 48 hpf, embryos were observed under a microscope. Phase-contrast images of the control-MO-injected (Ba,e) and Bad-MO [lower dose (0.1 mM)-injected; (Bb,f)] embryos show normal brain development. However, the embryos injected with higher doses of Bad-MO embryos show abnormal brain development ((Bc,g), 0.2 mM; (Bd,h) 0.4 mM at 24 and 48 hpf, respectively) in the hindbrain (HB), indicated by arrows. The defect rates (N, up to 120 embryos) were counted and are shown in Figure 3C,D for 24 and 48 hpf, respectively, revealing a statistically significant difference.