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Fig. 5 col15a1b−/− and tnc−/− embryos both display abnormal extrabranching phenotype. (A) whole-mount immunofluorescence of 27 hpf wild-type, col15a1b−/−, and tnc−/− mutant embryos with gfp antibodies (motor neurons, green). (B) Quantification of the axon length, nwt = 11 embryos, 55 axons, ncol15a1b−/− = 13 embryos, 65 axons, ntnc−/− = 13 embryos, 64 axons; (C) Distance between axons normalized to the distance between the two adjacent somites (in µm), nwt = 9 embryos, 25 somites, ncol15a1b−/− = 10 embryos, 31 somites, ntnc−/− = 10 embryos, 31 somites. Statistical analyses in B and C were performed using Tukey’s multiple comparisons test. Mean and SD are presented. (D) Whole-mount immunostaining of 27 hpf wt, tnc−/− and col15a1b−/− mutant embryos with anti-GFP to visualize individual motor axons (green) and their corresponding 3D representation using filament tracer (Imaris software). (E) Quantification of branching length normalized to axon length, nwt = 24 embryos, 118 axons, ncol15a1b−/− = 13 embryos, 64 axons, ntnc−/− = 13 embryos, 64 axons (F) branching point normalized to axon length, nwt = 21 embryos, 97 axons, ncol15a1b−/− = 13 embryos, 54 axons, ntnc−/− = 13 embryos, 56 axons. (E and F) Statistical analyses were performed using Kruskal–Wallis and Dunn’s multiple comparisons tests, median and range are represented. In all graphs, each point represents one motor axon. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All embryos are lateral views; anterior is left.