Both nerve branches contain lateral LMC axons in BmprIaflox/-hindlimbs. Comparison of anterior plexus axonal projection patterns in control and mutant embryos. A Lim1tlzallele was used to label lateral LMC axons with LacZ. (a,b) E11.75 control hindlimb; overlays of two adjacent sections. (a) NF+ nerves form three branches at the base of the hindlimb, with only the dorsal branch entering Lmx1b+ dorsal limb mesenchyme. (b) Only the dorsal limb branch contains LacZ+ lateral LMC axons. (c,d) E11.75 mutant hindlimb; in consecutive coimmunostained sections, dorsal, ventral and flank branches can be followed. (c) NF+ nerves form three branches, two of which enter Lmx1b+ limb mesenchyme. (d) Both limb nerve branches contain LacZ+ lateral LMC axons, while the ventral flank branch does not. (e) LacZ immunoreactivity was quantified and normalized for neurofilament immunoreactivity in each nerve branch; low-level signal in ventral limb and flank branches likely represents weak non-specific staining. Values are presented in relative units that represent the normalized LacZ signal in each branch. The relative signals in normal limbs (white bars) were (mean ± SEM): 5.3 ± 0.9 (dorsal), 1.1 ± 0.2 (ventral), 1 ± 0.1 (flank); n = 5 embryos. The relative signals in mutant limbs (black bars) were: 4.8 ± 1 (dorsal), 4.6 ± 0.9 (ventral), 1 ± 0.2 (flank); n = 5 embryos. P = 0.00005 for ventral limb branches, two-tailed t test. *P < 0.00005. D, dorsal limb nerve branch; F, ventral flank nerve branch; V, ventral limb nerve branch. Arrows: LacZ+ nerves. Dotted lines: limb outlines. Boxed areas in (a,c) are enlarged in (b,d).