|
Fig. 6 ColXV-B locally creates a soft microenvironment that elicits motor axon growth. (A) Confocal images of 27 hpf mnx1:gfp motor neurons cultivated on 5 or 40 kPa polyacrylamide hydrogels for 24 h. Zoomed images of the boxed areas are shown. (B) Left, initial image sample (Upper panel) and after image analysis using the customized version of “Angiogenesis Analyzer”, ImageJ software (Bottom panel). Asterisks, motor neuron cell bodies; arrows indicate master segments (orange); neurite branch (green); master junction (red). Right, quantification of the total master segment length and total branch length per cell, 24 h after motor neurons seeding (n = 418 cells for 40 kPa gels; n = 803 cells for 5 kPa gels). Triplicates of three independent experiments are represented. Statistical analysis was performed using a Student’s t-test. Mean and SEM are presented. ***P < 0.001. (C) Elastic modulus in the motor axon path obtained from cryosections of 27 hpf wt, col15a1b−/− and shh-injected embryos in mnx1:gfp background. Statistical analysis was performed using Kruskal–Wallis and pairwise Wilcoxon multiple comparisons tests. *P < 0.05. nwt = 1,342, ncol15a1b−/− = 690, nshh-injected = 400. Data are mean ± SEM