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G0 CRISPR targeting of Proteasome cluster genes leads to heart defects in zebrafish embryos and larvae. A.-C.hand2 G0 CRISPR leads to cardia bifida. A. 2 dpf control embryo showing myl7:EGFP heart. V, ventricle. A, atrium. Scale bar = 200 μm. B. 2 dpf hand2 G0 CRISPR embryo showing two domains (cardia bifida) of myl7:EGFP (arrows). C. Graph showing frequencies of different heart defect categories observed in G0 CRISPR embryos. Cardia bifida and heart looping defects were scored at 2 dpf. Hearts were again scored at 4 dpf for defects arising between 2 dpf and 4 dpf and were divided into two classes: lateral chamber arrangement and elongated chamber arrangement. Examples of the elongated chamber arrangement are illustrated in panels Q, S, T and V, and an example of the lateral chamber arrangement is shown in panel U. D. Graph showing frequencies of embryos with heart defects (at 4 dpf; all categories combined). Each dot represents an experimental replicate batch of embryos. Bars represent mean +/- SEM. N = 4-6 replicates per gene of interest; N = 24 control replicates. Each replicate batch consists of 7-75 embryos (mean = 36±16). The total number of embryos scored is given above the x-axis. These total numbers also apply to the corresponding columns in Panel C. ** P < 0.01. **** P < 0.0001. E.-O. Lateral views of 4 dpf larvae, showing representative G0 CRISPR phenotypes. The bubble (asterisk in E) is the swim bladder, an indicator of healthy larvae [40]. The arrowhead in I points to severe heart cavity swelling/edema. Scale bar = 500 μm. P.-V. Ventral views of hearts labeled with myl7:EGFP in 4 dpf larvae with pigment inhibited. Representative G0 CRISPR heart phenotypes are shown for each gene. Scale bar = 200 μm. V, ventricle. A, atrium.
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