Clustering of BCCs is perturbed in the absence of Sema6A and PlexinA1. (a-g) Longitudinal sections of HH25 spinal cords (as indicated by the dashed line in (a)) were stained with 1E8 (red) and anti-neurofilament antibodies (green) to analyze dorsal (b-d) and ventral (e-g) BCCs from untreated embryos (b, e) or embryos treated with dsRNA derived from SEMA6A (c, f) and PLEXINA1 (d, g), respectively. Dorsal BCC clusters in control embryos (b) were relatively homogenous in size, closely aligned with the roots, and regularly spaced. In contrast, in the absence of either Sema6A (c) or PlexinA1 (d), the size of BCC clusters was very variable and their arrangement was highly disorganized. Many axons were not in contact with BCCs at all or only with individual cells or microclusters (open arrowheads in (c, d)). Ventral BCC clusters were smaller than their dorsal counterparts even in control embryos (e). Therefore, the effect of Sema6A (f) or PlexinA1 (g) perturbation on cluster size was less obvious. However, the absence of Sema6A and PlexinA1 clearly disrupted the alignment of ventral BCC clusters (compare dashed lines in (e) with (f, g)). The color of the axons stained with anti-neurofilament antibodies and visualized with an Alexa350-coupled secondary antibody was changed to green using Adobe Photoshop CS2 to get better contrast. EGFP used to select the appropriate sections is not shown. MN, motoneurons; N, notochord. Bar 100 μm.