FIGURE 4.

Depletion of B56ε-s impairs midbrain/hindbrain boundary formation, without disrupting eye induction and eye field separation. A, whole mount in situ hybridization to show the expression of otx2, en2, wnt1, krox-20, and hoxB9 in stage 20 control embryos and embryos unilaterally injected with 10 ng of MO2. One of the dorsal animal blastomeres was injected at the 8-cell stage. Upper panels are control embryos. Lower panels are MO2-injected embryos. Right side was injected. B, whole mount in situ hybridization to show en2 expression in a stage 20 control embryo and embryos unilaterally injected with MO1 (5 ng), MO2 (10 ng), MO3 (10 ng), or MO4 (10 ng). Left side was injected. C, inhibitory effect of MO2 on the expression of en2 was rescued by ΔN. Upper panel shows en2 expression in a control embryo. Middle panel shows en2 expression in an embryo injected with MO2 (10 ng). Bottom panel shows en2 expression in an embryo injected with MO2 (10 ng) and ΔN (100 pg). Left side was injected. D, summary of results shown in C. Values represent percent of embryos with normal en2 expression. E, coinjection of ΔN (100 pg) rescued en2 expression in MO1 (5 ng)-injected embryos. Values represent percent of embryos with normal en2 expression. F, whole mount in situ hybridization showing en2 expression in a stage 20 control embryo and an embryo unilaterally injected with ΔN (100 pg). Left side was injected. G, whole mount in situ hybridization showing the expression of rx in control (top panels) and MO2-injected (bottom panels) embryos. Embryos were bilaterally injected at the 8-cell stage and harvested at the early neurula stage (left panels) and the early tadpole stage (right panels). In some of these experiments, nuclear β-galactosidase was coinjected as the lineage tracer.