Medial LMC axons innervate the BmprIaflox/-ventral flank mesenchyme. The columnar origin of neurons innervating the ventral flank mesenchyme at E13.5 was defined by HRP or RDA retrograde labeling. (a) Schematic of experiments shown in (b-d). (b) Flank nerves originate from medial LMC neurons in normal and mutant embryos, as Isl1+ medial LMC cells are readily HRP-labeled in triple coimmunostained sections. (c) Flank nerves in normal and mutant do not originate from medial MMC cells, as Lim3+ medial MMC cells are rarely HRP+ in triple coimmunostained sections. (d) Flank nerves in normal and mutant originate from medial LMC but not medial MMC as coimmunostained HRP+ Isl1+ FoxP1+ medial LMC cells were readily detected. In (b-d), upper panels are controls and lower panels are BmprIaflox/-mutants. Arrowheads indicate representative HRP+ neurons colabeled with nuclear markers and dotted lines in (b-d) indicate columnar division outlines. (e) Quantification of labeling data shows that axons projecting to ventral flank mesenchyme in normal and BmprIaflox/-mutant embryos are from Isl1+ Lim1- Lim3- FoxP1+ medial LMC neurons. The percentages of HRP+ or RDA+ cells were as follows. Isl1+ Lim1-: normal, 94 ± 1.2, n = 5 embryos, 648 neurons counted; mutant, 93 ± 2.1, n = 5, 388 neurons; P = 0.83. Lim3+: normal, 3.5 ± 1.8, n = 5, 515 neurons; mutant: 2.4 ± 1.4, n = 4, 283 neurons; P = 0.62. FoxP1: normal, 95 ± 1.5, n = 5, 479 neurons; mutant, 96 ± 1.5, n = 4, 262 neurons; P = 0.63). (f) Anterior-posterior length of the LMC is similar in normal and mutant E13.5 embryos, while the extent of the LMC labeled from the ventral flank is significantly increased in mutant embryos. LMC lengths (Isl1+/Hb9-GFP+ sections) were: normal, 1,658 ± 23 μm, n = 6 embryos; mutant, 1,690 ± 113 μm, n = 5; P = 0.745. Tracer extents (HRP+ or RDA+ sections) were: normal, 407 ± 96 μm, n = 5 embryos; mutant, 864 ± 116 μm, n = 5; P = 0.008.