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Fig. 3 The motor axon trajectory is formed by a central ColXV-B path delimited by TnC borders. (A) 3D Imaris images of whole-mount immunostaining of 27 hpf mnx1:gfp embryos with anti-rNC1 (magenta, ColXV-B), anti-TnC (cyan), and anti-GFP antibodies (green, motor neurons). Left panel, lateral views; Right panel, orthogonal views; anterior is left. The dotted line shows the horizontal myosepta. Asterisks indicate the vertical myosepta. The arrowhead points to the dorsal edge of the notochord. Numbers indicate the level of virtual orthogonal sections (B) Double immunostaining of 45 × 70 µm ColXV-B/TnC micropatterns with anti-rNC1 (magenta, ColXV-B) and anti-TnC (cyan), plot profile of ColXV-B and TnC staining (Fiji software). Micropattern with human native TnC (hnTnC) is shown. (C) Live confocal images of motor neurons 24 h after seeding on 31 × 70 µm ColXV-B/TnC micropatterns (Top panel) and ColXV-B control micropatterns (Bottom panel). (D) Quantification of the number of pixels corresponding to the neurites for each ColXV-B or TnC area expressed in percentage of the total micropattern area. Top panel, double ColXV-B/murine recombinant TnC (mrTnC) micropatterns; n = 30; Middle panel, ColXV-B/ hnTnC double micropatterns, n = 21; Bottom panel, ColXV-B control micropatterns, n = 23. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparisons tests. ****P < 0.0001; ns, not significant. Error bars are mean ± SEM.