Fig. 1

Epigenetic regulators in zebrafish dystrophic embryos.

A) Quantitative PCR analysis of p300/CBP transcript expression at 2 days post fertilisation (dpf) in control and morpholino-depleted embryos (MOdmd). (B) Confocal sections of Tg(actc1b:mCherry)^pc4 embryos immunostained for Dystrophin (green). Well organised pattern of muscle fibres (mCherry, red) and nuclei (DAPI, blue) in siblings, while heavily disrupted in dmd^{ta222a/ta222a} embryos (arrows), showing collapsed and wavy fibres which ends misalign with somatic borders (dashed lines). Overexpressing CBP (dmd^{ta222a/ta222a} + CBP; injection of 10⁷ RNA molecules of plasmid per embryo at one cell stage) has a clear positive effect on dystrophic embryos both at 2 and 6 dpf. (C, D) Frequency of dystrophic phenotype (see Materials and Methods) on 2 dpf Dystrophin-null embryos, either dmd^{ta222a/ta222a}mutants (C) or depleted with MOdmd (D). Overexpressing CBP (low = 10⁷, high = 10⁸ molecules of plasmid RNA injected per embryo) rescues the dystrophic phenotype of both dmd^{ta222a/ta222a}(C) and morphant (D; 10⁷ molecules) embryos as efficiently as blocking HDACs (TSA), even at lower concentrations (C,D). A mutated CBP form lacking the HAT domain (ΔHAT; 10⁷ molecules) significantly decreases the rescue efficiency (C). Individual values for independent experiments are plotted, bars represent the average ± SD and boxes above plots indicate total number of embryos analysed. (E) Representative confocal sections of 2 dpf embryos immunostained for Dystrophin (green) to confirm Dystrophin presence in siblings and depletion in MOdmd embryos, counterstained with DAPI (blue). Both TSA (200 nM) and CBP (10⁷ molecules) are able to rescue the muscle phenotype of MOdmd embryos, while ΔHAT injected embryos most often show muscle damage (10⁷ molecules). nt = neural tube. (F,G) Quantitative PCR analysis of crebbp, p300 and follistatin endogenous transcripts expression at 2 dpf. See text for details on ANOVA (A,F,G) or T tests (B,C), performed with cut-offs: (*) < 0.05, (**) < 0.005, (***) < 0.001, (****) < 0.0001. ns = non significant.