Figure 5

Quantitative TUNEL analysis of cell death: knockdown, co-knockdown, and antagonism studies. Cell death levels were measured in brd2bMO morphants and control embryos under various treatments using fluorescence TUNEL assay followed by quantitative laser-scanning confocal microscopy. The number of apoptotic nuclei in multiple optical sections from the brains of three to ten embryos per treatment, depending on experiment, were compared (see Methods for details). (A) RNA rescue treatments: brd2bMO1-injected (2bMO1), human Brd2 RNA-injected (HsRNA), brd2bMO1 + human Brd2 RNA-injected (2bMO+HsRNA), and uninjected (control). Means vary significantly by treatment (p < 0.0001, one-way ANOVA, Tukey?s HSD), with the greatest difference between 2bMO1 and all other treatments and no significant difference between ?control? and ?2bMO+HsRNA?, indicating effective rescue by exogenous human Brd2 RNA. Cell death numbers for HsBrd2 RNA-injected controls were obtained from embryos from a separate clutch but reflect what we have consistently seen over multiple independent trials. (B) brd2bMO knockdown corroboration treatments: brd2bMO1-injected (2bMO1), brd2bMO2-injected (2bMO2), Crispr-Cas9-brd2b disruption (Crispr2b), and uninjected (control). Means vary significantly by treatment (p < 0.0001, one-way ANOVA, Tukey HSD), with Crispr2b, 2bMO1 and 2bMO2 all showing effects significantly greater than ?control?; 2bMO1 and 2bMO2 show equivalent and moderate effects, while Crispr2b shows the greatest effect. Thus, three independent treatments targeting brd2b result in similarly increased cell death levels, showing gene-specificity of effect. (C) p53-dependent off-target effect treatments: brd2bMO1-injected (2bMO1), brd2bMO1 + p53MO co-injected (2bMO1 + p53MO), uninjected (control), and p53MO-injected (p53MO). Means vary significantly by treatment (p < 0.0001, one-way ANOVA, Tukey?s HSD), with no significant difference between 2bMO1 and ?2bMO1 + p53MO? or between ?control? and p53MO, but significant differences between the two pairs, indicating excess apoptosis is brd2b-specific, and not the result of p53-dependent off-target effects. (D) Co-knockdown treatments: brd2aMO1-injected (2aMO1), brd2bMO1-injected (2bMO1), brd2aMO1 + brd2bMO1 co-injected (2aMO+2bMO), and uninjected (control). Means vary significantly by treatment (p < 0.0001, one-way ANOVA, Tukey?s HSD), with ?control? and ?2aMO + 2bMO? not significantly different from each other and both significantly different from either 2bMO1 or 2aMO1; 2bMO1 shows a greater effect than 2aMO1. Simultaneous knockdown of both paralogs suppresses excess cell death observed in single knockdowns of either paralog and restores wild type levels of apoptosis. Panels (E?H) Rescue-Enhancement studies using zebrafish brd2a and brd2b-L in vitro-synthesized RNAs (not human Brd2) tested functional antagonism between paralogs at the level of apoptosis. (E): brd2bMO rescue treatments: brd2bMO2 (2bMO2), brd2b-LRNA (2bRNAL), brd2bMO2 + brd2b-LRNA (2bMO2 + 2bRNAL), and uninjected (control). Means vary significantly by treatment (p-0.0286, one-way ANOVA, Tukey?s HSD), with ?2bMO2+2bRNAL? closer to ?control? in effect (p = 0.8407) than either 2bMO1 (p = 0.1319) or 2bRNAL (p = 0.0295), indicating partial rescue of the brd2bMO morphant defect. (F) brd2bMO enhancement treatments: brd2aRNA-injected (2aRNA), brd2bMO2-injected (2bMO2), brd2aRNA + brd2bMO2 co-injected(2bMO2+2aRNA), and uninjected (control). Means vary significantly by treatment (p = 0.0008, one-way ANOVA, Tukey?s HSD), with ?2bMO2+2aRNA? giving significantly greater effect compared to ?control? (p = 0.0009) than either 2bMO2 (p = 0.2405) or 2aRNA (p = 0.9151). (G) brd2aMO rescue treatments: brd2aMO1-injected (2aMO1), brd2aRNA-injected (2aRNA), brd2aMO1 + brd2aRNA co-injected (2aMO1 + 2aRNA), and uninjected (control). Means vary significantly be treatment (p = ?0.0176, one-way ANOVA, Tukey?s HSD), with ?2aMO1 + 2aRNA? closer to ?control? in effect (p = 0.9985) than either 2aRNA (p = 0.3286) or 2aMO1 (p = 0.0.0294), indicating partial rescue of the brd2aMO morphant defect. (H) brd2aMO enhancement treatments: brd2aMO1-injected (2aMO1), brd2b-LRNA-injected (2bRNAL), brd2aMO1 + brd2b-LRNA co-injected (2aMO1 + 2bRNAL), and uninjected (control). Means vary significantly by treatment (p = 0.0084, one-way ANOVA, Tukey?s HSD), with ?2aMO1+2bRNAL? giving significantly greater effect compared to ?control? (p = 0.0085), than either 2aMO (p = 0.3433) or 2bRNAL (0.9342). Note: for experiments in A, B, and D, apoptotic cell counts were obtained from 55 to 60 optical sections from 6 to 10 embryos per treatment; for experiment C, from maximum projection images from 3 embryos per treatment; for experiments (E?H), from 35 optical sections from 3 to 5 embryos per treatment. Green diamonds: confidence interval (95%) for group means with standard error. Black line: grand sample mean. Black circles: comparison circles for absolute differences of group means.