Knockout of sema6D caused both aberrant phenotypes in PMNs and ISVs. (A) Schematic diagram showing the targeting site of the gRNA on the sema6D gene. Starting codon (ATG) site is indicated by arrow. (B) The statistics of the ratio of wildtype to mutant sequences (n = 50). (C) Knockout patterns of sema6D-gRNA/cas9-injecting embryos by sequencing. (D) Confocal imaging analysis of primary motor neurons and intersegmental vessels in control and the F0 generation of the injected Tg(mnx1:EGFP::kdrl:ras-mCherry) zebrafish at 48 hpf, blue arrowheads indicate aberrant PMN and ISV. (E) The statistical analysis of the ratio of aberrant axonal projection of Caps (P = 0.0006) and short Caps (P = 0.0006) in the wild, F0 knockouts, and cas9 negative control at 48 hpf (n = 7). About 9–11caps were used for statistics per zebrafish. Mann-Whitney test. Values with *** above the bars are significantly different (P < 0.001). (F) The statistical analysis of the ratio of vessels that span different segments (P = 0.0006) and the ratio of proliferated vessels (P = 0.006) in the wild, F0 knockouts, and cas9 negative control at 48 hpf (n = 7). About 9–12 ISVs were used for statistics per zebrafish. Mann-Whitney test. Values with *** above the bars are significantly different(P < 0.001).
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