Figure 2

Skeletal muscle cell-specific targeting of foxm1 with CRISPR/Cas9 is detrimental. (A) Expression of foxm1 in whole zebrafish embryo (except skeletal myofibers) and FACS-sorted skeletal myofibers at 3 dpf. Values are mean ± SD from n = 6. **** = p < 0.0001 by Student’s t-test. (B) Representation of the Tol2 cassettes used for transgenesis in this study. White bar represents the Tol2 recombination sites, pA refers to the poly-A sequence and U6 refers to the ubiquitous U6 promoter. (C) Embryo injected with mylpfa:mCherry, at 1 (top) and 5 dpf (bottom). Fluorescence microscopy images correspond to the embryo areas indicated by red boxes on the right. (D) Quantification of mCherry-positive (Control) or the GFP-positive (Cas9 and Cas9/sgRNA) cells at 1, 2, 3 and 5 dpf. Values are mean ± SD from n ≥ 34 embryos, * = p < 0.05, ** = p < 0.01, **** = p < 0.0001 by one-way ANOVA with Bonferroni correction for multiple comparisons. (E) Embryo injected with mylfpa:Cas9-T2A-GFP (Cas9) and mylfpa:Cas9-T2A-GFP;U6:8.2 (Cas9/sgRNA), at 1 (top) and 5 dpf (bottom). (F) 2 dpf embryos injected with mylpfa:mCherry (Control), mylfpa:Cas9-T2A-GFP (Cas9) or mylfpa:Cas9-T2A-GFP;U6:8.2 (Cas9/sgRNA) stained with anti-cleaved caspase-3 (Cc3) antibody. (G) Quantification of 2 dpf embryos with Cc3-negative (Cc3−, gray) and Cc3-positive (Cc3+, red) myofibers from n ≥ 42 embryos per condition. **** p < 0.0001 by Fisher’s exact test using the discrete number of embryos per condition, comparison with control embryos. Scale bar: 100 µm.