Figure 4

Brd2b knockdown increases cell death in the CNS of prim 5 morphant embryos but reduces cell death in the cloaca of the pronephros. Dark-field images of representative 24 hpf prim5 embryos treated as indicated and after TUNEL assay for apoptotic nuclei. Panels (A?D) (heads and trunks) show treatment groups for testing gene-specificity of apoptotic effects: (A) uninjected; (B) brd2bMO1-injected; (C) brd2bMO1 + HsBrd2RNA-injected; and (D) Crispr-Cas9-brd2b-disrupted embryos. HsBrd2RNA co-injection rescues, while Crispr-Cas9 treatment phenocopies, excess apoptosis in the brain and trunk overall of morphants, showing effects are specific to brd2b. Arrows indicate specific regions of reduced apoptosis at the cloaca (upper arrows) and increased apoptosis in the PBI (lower arrows) in the ventral trunk of morphant and Crispr-Cas9-treated, but not control or rescued embryos. Panels (E?H) (heads and trunks) show treatment groups for testing p53-dependent off-target effects: (E) uninjected; (F) p53MO-injected; (G) brd2bMO1-injected; and (H) brd2bMO1? + p53MO co-injected. p53MO co-injection does not abrogate excess apoptosis in morphants, ruling out off-target effects as the cause of cell death. See Figure 5 for quantitative TUNEL data from these studies. See Supplementary Figure S2 for images on HsBrd2 RNA control embryos.