Figure 3

Brd2b knockdown results in reduced hindbrain, ill-defined MHB region, and trunk abnormalities similar to Brd2a morphants, but presents unique circulatory and pronephric defects. Brightfield images of 24 hpf prim 5 embryos (A?F), and larvae at 6 days (H?J). Lateral views of head and trunk of representative embryos from indicated treatment groups: (A) uninjected; (B) brd2bMO1-injected; (C) Brd2bMO1 + HsBrd2RNA co-injected; (D) Brd2MO2-injected; (E) Crispr-Cas9-brd2b-disrupted; and for comparison, (F) paralog brd2aMO-injected. In A) mhb = midbrain-hindbrain; pd = pronephric duct; c = cloaca; pbi = peripheral blood island. Head and trunk defects in morphants and Crispr-Cas9-treated embryos are indicated by black arrows, and include reduced brain and ill-defined MHB region, misformed pronephric duct, and disorganized PBI. These defects are substantially rescued by co-injection of human Brd2 RNA, recapitulated by a non-overlapping anti-brd2b morpholino (MO2), and phenocopied by Crispr-Cas9 disruption, showing gene-specificity of observed effects. (G) Crispr-Cas9 brd2b target validation, showing intact PCR products from wildtype (lane 1, wt) and Crispr-disrupted (lane 2, 2b) brd2b locus; wildtype PCR product treated with mismatch-detecting resolvase (lane 4, wt+) and untreated (lane5, wt?); Crispr-disrupted PCR product treated with resolvase (lane 6, 2b+) and untreated (lane 7, 2b?). Crispr-disrupted brd2b locus treated with resolvase shows cleavage products (asterisk highlighting lane 6), while wildtype locus remains intact, indicating that Crispr-Cas9 targeted designated sequences in the brd2b locus. Pixel density of the 600 bp intact product band is reduced from 980 in untreated (lane 7) to 528 in treated (lane 6) Crispr-disrupted embryos, indicating close to 50% efficacy (see Methods). (H) Six day old morphant larvae injected at 2 cell stage with 1 ng brd2bMO1, or (I,J) 2ng brd2bMO1, illustrating dosage-dependent severe heart edema and trunk deformities, due to nearly complete lack of blood circulation. See Table 1 for population morphology data from these studies. See Supplementary Figure S1 for HsBrd2 RNA control embryos images.