Wfs1b regulates M-cell axon regeneration in vivo. a Hybridization of the transgenic line: Tg (Tol 056: EGFP) and wfs1b mutants were crossed for two consecutive generations to obtain Tg (Tol 056: EGFP)/ wfs1b +/- and Tg (T056: EGFP); wfs1b −/− lines. b Representative images of the M-cell axon before and after ablations by a two-photon laser. Asterisk, injury site; arrowhead, cloacal pore; scale bar, 50 μm. c, d Confocal imaging of M-cell axons between wfs1b+/+ and wfs1b−/− groups at 8 dpf and the regeneration length at 2 dpa. Violin plot shows all data points, including minimum, maximum, median, and quartiles. Scale bar, 20 μm, P < 0.0001, control, n = 24; wfs1b−/−, n = 26. Assessed by unpaired t test. e, f Representative images of embryos from the wildtype and the mutant at 6 dpf (scale bar, 500 μm), and measured total body length from 4 to 6 dpf before axotomy (4 dpf, wildtype: 3.603 ± 0.01402 cm, wfs1−/−: 3.549 ± 0.02201 cm, P = 0.1708; 5 dpf, wildtype: 3.745 ± 0.01504 cm, wfs1−/−: 3.727 ± 0.01679 cm, P = 0.8255; 6 dpf, wildtype: 3.790 ± 0.01691 cm, wfs1−/−: 3.806 ± 0.01569 cm, P = 0.8842; n = 30). Assessed by two-way ANOVA. ns, not significant. g, h Defined lengths of M-cell axons from the cloaca to the end were not notably different among WT, homozygous, and heterozygous larvae (wfs1b+/+: 1042 ± 19.51 μm; wfs1b+/-: 985.2 ± 21.47 μm, P = 0.0689; wfs1b−/−: 995.6 ± 21.98 μm, P = 0.1363; n = 9). Assessed by ordinary one-way ANOVA/Tukey’s multiple-comparisons test (wfs1b+/+ versus wfs1b+/-: P = 0.1594; wfs1b+/+ versus wfs1b−/−: P = 0.2856; wfs1b+/- versus wfs1b−/−: P = 0.9342) White asterisk: ablation point. Scale bar, 20 μm. ns, not significant
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