Figure S4.
Details of the CRISPR/Cas9 F0 mutant screen for PS receptors in zebrafish. (A) Workflow of F0 mutant genotyping. Target regions were amplified from genomic DNA. Gene knockout in F0 mutants differed from controls by loss of a unique restriction site. gRNA efficiency was estimated from the ratio of undigested to digested bands on the agarose gel. (B) Agarose gels show genotyping results of bai1, tim1, and axl in the bai1+tim1+axl mutants and tyr controls. P, PCR product. (C) Sanger sequencing of bai1, tim1, and axl target sites in mutant and control animals, showing frameshift mutations in the mutants (KO). Arrows indicate gRNA position. Dashed lines mark Cas9 cleavage sites. (D) gRNA efficiencies in the triple F0 mutant screen (n = 5 larvae). (E) gRNA efficiencies in the double and single F0 mutant screen (n = 8 larvae). (F and G) Average volume of myelin fragments within individual microglia in triple F0 mutants (F), and double and single mutants of bai1 and tim1 (G). n = 5 larvae in F, n = 7 larvae in bai1+tim1, n = 8 larvae for tyr, bai1, and tim1 in G. One-way ANOVA with post-hoc Tukey’s test: tyr vs. tim4+bai1+mertka: P = 0.0002, tyr vs. tim4+mertka + axl, tyr vs. bai1+axl + tim1, tyr vs. mertka + tyro3+bai1 and tyr vs. mertka + axl + tyro3: P < 0.0001, tyr vs. bai1+tim1: P = 0.0160, all other comparisons in F and G were non-significant. (H−J) Microglia in the spinal cord of control and F0 mutant zebrafish larvae in the PS receptor KO screen. Example images (H) and quantification of the number of microglia in triple mutants (I) and double and single mutants of bai1 and tim1 (J). n = 5 larvae in I, n = 7 larvae for bai1+tim1, n = 8 larvae for tyr, bai1, and tim1 in J. One-way ANOVA with post-hoc Tukey’s test: tyr vs. bai1+tim1: P = 0.0026, bai1+tim1 vs. tim1: P = 0.0262, all other comparisons in I and J were non-significant. (K−M) Quantifications of number of OPCs in Tg (olig1:memEYFP) (K; n = 11 larvae), number of oligodendrocytes (L; n = 13 larvae), and sheath lengths in Tg (mbp:EGFP-CAAX) (60–65 individual myelin sheaths from n = 3 fish; M) in the dorsal spinal cord of 10 dpf control larvae injected with a three scrambled gRNAs and bai1;axl;tim1 F0 larvae. Two-sided Student’s t test: P = 0.8874 (K), P = 0.3769 (L), P = 0.8051 (M). Data represent means ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Scale bar: 20 µm (H). Source data are available for this figure: SourceData FS4.

Details of the CRISPR/Cas9 F0 mutant screen for PS receptors in zebrafish. (A) Workflow of F0 mutant genotyping. Target regions were amplified from genomic DNA. Gene knockout in F0 mutants differed from controls by loss of a unique restriction site. gRNA efficiency was estimated from the ratio of undigested to digested bands on the agarose gel. (B) Agarose gels show genotyping results of bai1, tim1, and axl in the bai1+tim1+axl mutants and tyr controls. P, PCR product. (C) Sanger sequencing of bai1, tim1, and axl target sites in mutant and control animals, showing frameshift mutations in the mutants (KO). Arrows indicate gRNA position. Dashed lines mark Cas9 cleavage sites. (D) gRNA efficiencies in the triple F0 mutant screen (n = 5 larvae). (E) gRNA efficiencies in the double and single F0 mutant screen (n = 8 larvae). (F and G) Average volume of myelin fragments within individual microglia in triple F0 mutants (F), and double and single mutants of bai1 and tim1 (G). n = 5 larvae in F, n = 7 larvae in bai1+tim1, n = 8 larvae for tyr, bai1, and tim1 in G. One-way ANOVA with post-hoc Tukey’s test: tyr vs. tim4+bai1+mertka: P = 0.0002, tyr vs. tim4+mertka + axl, tyr vs. bai1+axl + tim1, tyr vs. mertka + tyro3+bai1 and tyr vs. mertka + axl + tyro3: P < 0.0001, tyr vs. bai1+tim1: P = 0.0160, all other comparisons in F and G were non-significant. (H−J) Microglia in the spinal cord of control and F0 mutant zebrafish larvae in the PS receptor KO screen. Example images (H) and quantification of the number of microglia in triple mutants (I) and double and single mutants of bai1 and tim1 (J). n = 5 larvae in I, n = 7 larvae for bai1+tim1, n = 8 larvae for tyr, bai1, and tim1 in J. One-way ANOVA with post-hoc Tukey’s test: tyr vs. bai1+tim1: P = 0.0026, bai1+tim1 vs. tim1: P = 0.0262, all other comparisons in I and J were non-significant. (K−M) Quantifications of number of OPCs in Tg (olig1:memEYFP) (K; n = 11 larvae), number of oligodendrocytes (L; n = 13 larvae), and sheath lengths in Tg (mbp:EGFP-CAAX) (60–65 individual myelin sheaths from n = 3 fish; M) in the dorsal spinal cord of 10 dpf control larvae injected with a three scrambled gRNAs and bai1;axl;tim1 F0 larvae. Two-sided Student’s t test: P = 0.8874 (K), P = 0.3769 (L), P = 0.8051 (M). Data represent means ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Scale bar: 20 µm (H). Source data are available for this figure: SourceData FS4.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal