Injection of gbx2 morpholino induces anterior hindbrain cell death. A–Q: Effect of gbx2-MO, p53-MO, and gbx2-MO/p53-MO co-injection on embryos. A–H: Lateral view, anterior to the left of embryos stained with acridine orange and viewed by fluorescent microscopy at 10-somite (A–D), 20-somite (E–H), and 24 hpf (inset in E–H) showing increased cell death in r2–r5 at 20-somites and r2, r3, and r5 at 24 hpf in gbx2 morphants. I–P: Dorsal views, with anterior to the left. I–L: Two-color in situ hybridization examining the expression of pax2.1 (black) and krox20 (red) at 20-somite stage, shows anterior hindbrain truncation in gbx2 morphants. L: Co-injection of gbx2-MO/p53-MO slightly reduces anterior hindbrain truncation. M–P: GFP DAB antibody staining of 36-hpf embryos examining cell body clusters. Q: Distance between posterior midbrain and r3. Arrows indicate disruption of distinct nV clustering in r2 and r3. *, Position of r4; **P < 0.0001 (Student′s t-test); hpf, hours post-fertilization; MO, morpholino; r, rhombomere; s, somite stage.
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