Medial LMC axons in the BmprIaflox/- mutant are redirected from the ventral limb into the ventral flank. Scip immunostaining and retrograde labeling (HRP or RDA) of nerves at E13.5 was used to define the contribution of Scip+ neurons to each nerve branch. (a) Retrograde labeling from normal dorsal or ventral limb mesenchyme. Dorsal nerves originate from Scip- lateral LMC neurons (a, i-vi). HRP-labeled cells do not express Scip and are Isl1- Lim1+ lateral LMC cells (white arrows). Scip+ medial LMC cells contribute to ventral nerves (a, vii-xii). Many HRP-labeled cells express Scip and all Scip+ cells are Isl1+ Lim1- medial LMC neurons (white arrows). (b) Retrograde labeling from normal or mutant ventral flank mesenchyme. In normal and mutant embryos retrogradely labeled Scip- cells are readily detected in sections of rostral spinal cord (b, i-vi). Labeled cells are detected in caudal lumbar spinal cord only in BmprIaflox/- mutants (b, vii-xii, white arrows). Many of these labeled neurons are also Scip+. Similar staining patterns were observed in three embryos for each labeling experiment. Representative sections are shown of labeled motor columns co-immunostained for HRP and Scip. Experiments in (a,b) are diagrammed schematically to the right, and boxed areas show the regions of the images. (c) Quantification of flank retrograde labeling data. The fraction of HRP+ or RDA+ flank-labeled cells that are also Scip+ differs significantly (P < 0.02) between normal and mutant (normal: white bar, 6.4% ± 2.6%, n = 6 embryos, N = 1,018 labeled neurons; mutant: black bar, 23.6% ± 4.7%; n = 5 embryos, N = 784 labeled neurons). (d) The anteroposterior span of the Scip+ pool within the E13.5 lumbar LMC does not differ significantly between normal and mutant embryos (normal: 701 ± 26 μm, n = 6 embryos; mutant: 592 ± 94 μm, n = 5; P = 0.219).