Fig. 6

The cell death phenotype of brd2aMO morphants is rescued by co-injection of human Brd2 RNA, and is recapitulated by treatment with Brd2 BET inhibitor.

Co-injection studies were conducted with 4 ng brd2aMO1and 250 pg in vitro transcribed human Brd2 RNA (A–J) or 2 ng brd2aMO1 and 4 ng p53MO (K–R) using a total of at least three clutches and 40–60 embryos per treatment. Embryos at prim 5–6 were assayed for cell death using TUNEL and analyzed with either laser scanning confocal (A–J) or conventional fluorescence (K-R) microscopy. Embryos challenged with 1 μM Brd2-specific BET inhibitor (BIC1) starting at 3 hpf are also shown (S,T). Light and dark field images of representative embryos from RNA rescue studies are shown for uninjected wildype (A,B), RNA-injected (C,D), brd2aMO1-injected (mild E,F and moderate G,H), and brd2aMO1 + RNA co-injected (I,J) groups. Representative embryos from p53MO rescue studies are shown for uninjected wildtype (K,L), p53MO-injected (M,N), brd2aMO1-injected (O,P), and brd2aMO1 + p53MO co-injected (Q,R) groups. For Brd2 BET inhibition, a total of 30 embryos and three clutches per treatment were used. Representative embryo at prim 5 after 1 μM BIC1 inhibitor treatment from 3 hpf is shown in S,T. Co-injection with human Brd2 mRNA improves brain morphology significantly and reduces cell death in morphants to levels close to normal (A–D and I, J vs. E–H). In contrast, co-injection with a 4:2 ng ratio of p53MO:brd2aMO1 shows only slight improvement in brain morphology and in levels of cell death (O,P vs. Q,R and E–H vs. I,J). Brd2 inhibitor BIC1 treatment phenocopies brd2aMO cell death, independent of morpholino use. Refer to Fig. 8A,B,C for quantitative analysis.