Fig. 4: Editing of candidate genes affects embryonic heart rate in medaka embryos.

A Validation workflow including zygotic microinjections using a HdrR (myl7::eGFP; myl7::H2A-mCherry) reporter line, high-throughput image-based heart rate quantification in normally developed injected specimens at 4 days post fertilization (dpf). (B, C) Heart rate distributions and absolute heart rate differences of morphologically normal crispants (n = 13 to 22 embryos) and editants (n = 4 to 22 embryos) compared to mock-injected control embryos (n = 8 to 12 embryos) at 21 °C, 28 °C, and 35 °C with CRISPR-Cas9- (B) and base editor-mediated (C) targeted mutagenesis of candidate genes (heart rate values are listed in Data S2 and sample numbers in Data S3). The significance of heart rate differences between the edited group and its corresponding control was assessed with the two-sided Wilcoxon test; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (p values listed in Data S4). Data is visualized as box plots (median+/− interquartile range between the 25th and 75th percentiles) and overlaid scatter plots of heart rate measurements; knockouts of adprhl1, btbd1, blzf1, phka2, and rrad have temperature-dependent effects on heart rate. Schemes are adapted from a previous publication²⁰ under a CC BY 4.0 Creative Commons licence.