Figure 8.

Only lateral LMC axons innervate BmprIa^flox/- hindlimb mesenchyme. HRP retrograde labeling at E13.5 identified spinal cord neurons that contribute to each limb nerve branch. (a) Dorsal limb nerve originates from the lateral LMC in normal and mutant as HRP+ Lim1+ lateral LMC cells are readily detected, while HRP+ Isl1+ cells are not, in triple co-immunostained sections. (b) Schematic of the experiment in (a). (c) Ventral limb nerve originates from the medial LMC in normal embryo as HRP+ cells are also Isl1+ Lim1-. In contrast, this nerve originates from lateral LMC neurons in the mutant as HRP+ cells are Lim1+ Isl1-. (d) Schematic of the experiment in (c). In (a-d) the upper panels show control embryos and the lower panels show BmprIa^flox/- mutants. Arrowheads indicate representative HRP+ Lim1+ or HRP+ Isl1+ neurons. Dotted lines in (a,c) indicate lateral and medial LMC outlines. Boxed areas in (b,d) indicate regions of interest in (a,c). (e,f) Quantification of (e) dorsal and (f) ventral retrograde labeling data. Lim1+ Isl1- HRP+ lateral LMC or Lim1- Isl1+ HRP+ medial LMC cells were counted in triply immunostained sections after retrograde labeling of control and BmprIa^flox/- hindlimbs, and are shown as percentages of total HRP+ cells. Dorsal limb HRP injection does not label significantly different populations in normal and mutant (normal: lateral LMC 97% ± 3%, n = 3 embryos, N > 150 HRP+ neurons; mutant: lateral LMC 95% ± 2%, n = 8 embryos, N > 350 HRP+ neurons; P = 0.6). Ventral limb HRP injection does label significantly different cell populations (normal: medial LMC 95% ± 1%, n = 6 embryos, N > 300 HRP+ neurons; mutant: medial LMC 6% ± 2%, n = 9 embryos, N > 400 HRP+ neurons; P < 0.00001). *P < 0.00001.