Figure 2. Paxillin function is required for the selection of limb axon trajectory. All images are from chick HH st. 28/29 lumbar levels. A–C, Detection of Foxp1, Isl1, GFP, and paxillin in LMC neurons of chick embryos electroporated with GFP (A), scrambled [paxillin]siRNA and GFP (B), or [paxillin]siRNA and GFP (C). D–F, Detection of Foxp1, paxillin, and GFP in dissociated LMC neurons purified from chick embryos electroporated with GFP (D), scrambled [paxillin]siRNA and GFP (E), or [paxillin]siRNA and GFP (F). G, Quantification of effects of GFP, scrambled [paxillin]siRNA, and GFP, or [paxillin]siRNA + GFP electroporation on paxillin mRNA levels. The e/u ratio was obtained in at least 15 sections of five embryos. H–J, Detection of Isl1, Foxp1, and GFP proteins and mouse paxillin mRNA in the LMC of chick HH st. 28/29 electroporated with GFP (H), paxillin and GFP (I), or paxillinY31F, Y118F and GFP (J) expression plasmids. Mouse (m) paxillin probe designed to specifically recognize ectopic mouse paxillin mRNA following m paxillin electroporation. K, Quantification of effects of GFP, scrambled [paxillin]siRNA and GFP, [paxillin]siRNA and GFP, paxillin and GFP, or paxillinY31F, Y118F and GFP electroporation on axon outgrowth. The ratio of the length of GFP+ LMC axons of each experiment to the GFP control group were obtained in at least 15 sections of five embryos. L, Number of LMC motor neurons expressed as the average number of total (Foxp1+) LMC neurons per section (# Foxp1+ MNs/section). Numbers of embryos: n = 5 for all groups. M, N, Number of total or electroporated medial (Foxp1+ Isl1+) and lateral (Foxp1+ Isl1–) LMC motor neurons in lumbar spinal cord expressed as the percentage of total motor neurons [Foxp1+ MNs (%); M] or electroporated motor neurons [GFP+ MNs (%); N]. Numbers of embryos: n = 5 for all groups. O–U, Neurofilament and GFP detection in the limb nerve branches in the crural plexus of chick embryos electroporated with the following expression plasmids and siRNAs: GFP (O), scrambled [paxillin]siRNA and GFP (P), [paxillin]siRNA and GFP (Q), paxillin and GFP (R), [paxillin]siRNA, mouse paxillin and GFP (S), medial LMC axonal marker, e[Isl1]::GFP (T), or [paxillin]siRNA and e[Isl1]::GFP (U). Quantification of GFP signals in all electroporation experiments expressed as, respectively, percentage in dorsal and ventral limb nerves [GFP Fluo (%)]. Numbers of embryos: n = 5 for all groups. d, Dorsal; v, ventral. n.s. = not significant; ***p < 0.001; **p < 0.01; statistical significance computed using Fisher's exact test (A–N) or Mann–Whitney U test (O–U); solid lines in each scatterplot show the median difference. Scale bars: A–C, H–J, 45 μm; D–F, 35 μm; O–U, 150 μm.