Medial LMC axons enter only the ventral flank nerve branch in the BmprIaflox/-hindlimb. Anterograde labeling was used to determine the trajectories of axons originating within the medial LMC. (a) Schematic showing the dorsal approach used for the anterograde HRP labeling of spinal cord neurons at E12.5. Regions of interest in (b,c) are boxed. (b) Triple co-immunostaining showing spinal cords in which the vast majority of HRP+ cells are Isl1+ Lim1- medial LMC neurons. (c) In normal hindlimbs, both ventral flank and ventral limb branches are labeled with HRP (n = 5 embryos, label detected in ventral flank branch = 5/5, ventral limb branch = 5/5, dorsal limb branch = 0/5), while neurofilament labeling identifies all three nerve branches. In mutant hindlimbs, only the ventral flank branch is HRP+ (n = 5 embryos, ventral flank branch = 5/5, ventral limb branch = 0/5, dorsal limb branch = 0/5). Adjacent sections from anterior plexus are shown for both normal and mutant. Arrowheads: representative HRP+ Isl1+ neurons. Arrows: HRP+ nerve branches. Dotted lines: outlines of lateral and medial LMC. D, dorsal limb nerve branch; F, ventral flank nerve branch; V, ventral limb nerve branch.