Fmn2b knock-down impairs axonal outgrowth in the spiral fiber tracts. Whole-mount immunostaining using anti-neurofilament antibody 3A10 stains axons in 96-hpf zebrafish hindbrain (A–C, E–G). A, Spiral fiber tracts are marked with red arrowheads in control morphants. B, Fmn2b knock-down using splice blocking morpholino (MO SB Fmn2b; 2 ng/embryo injected in the cytoplasm) reveals defects in the spiral fiber tract outgrowth. The spiral fiber defect is recapitulated with the translation blocking morpholino MO TB Fmn2b in a dose-dependent manner (Extended Data Fig. 5-1A,B). C, The phenotype in Fmn2b morphants could be rescued by injection of 300 pg mFmn2-GFP mRNA in the MO SB Fmn2b-injected embryos at one cell stage. Scale bar: 20 μm. D, A total of 48% Fmn2b morphants (n = 31) show the absence of spiral fiber tracts as compared with none in the control morphants (n = 27). However, only 5% of the embryos rescued with mFmn2-GFP mRNA (n = 40) showed the defects. A χ2 test of independence was performed to compare the three groups, χ2 (df = 2) = 95.75, p < 0.0001. E, Spiral fiber neurons marked with red arrowheads in embryo injected with Cas9 mRNA. F, Fmn2b knock-down using 30-pg dose of each of the two sgRNAs against fmn2b causes defects in the outgrowth of the spiral fiber tracts in Fmn2b crispants. G, Fmn2b knock-down using 100-pg dose of each of the two sgRNAs against fmn2b causes defects in the outgrowth of the spiral fiber tracts in Fmn2b crispants. Scale bar: 20 μm. H, Quantification of the phenotype reveals that Fmn2b crispants injected with 30-pg sgRNAs and Cas9 mRNA (n = 25) show absence of both the spiral fiber tracts in 40% embryos and thinning or absence of only one tract in 44% embryos, as compared with no defects observed in the group injected with the Cas9 mRNA only (n = 27). Fmn2b crispants injected with 100-pg sgRNAs and Cas9 mRNA (n = 28) show an increase in the severity of the phenotype with 67.85% embryos showing absence of both tracts, 32.15% showing thinning or single tract and no embryos with both the spiral fiber tracts intact. A χ2 test of independence was performed to compare the three groups, χ2 (df = 4) = 255, p < 0.0001. Representative images of the single spiral fiber tract outgrowth and thinning of the spiral fiber tract in Fmn2b crispants are included in the Extended Data in comparison to Cas9 mRNA-injected embryos (Extended Data Fig. 5-1C–E). Crispants showed similar morphologic defects during early development when compared with morphants (Extended Data Fig. 5-1F). Various indels and base changes introduced in crispants by co-injection of sgRNA-1 and sgRNA-2 along with Cas9 mRNA are summarized in Extended Data Figure 5-2A,B.
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