Nogo-A KO mouse embryos show defects in the development of fore- and hindlimb innervation. (A) Expression of Nogo-A during mouse spinal cord development shown by in situ hybridization. Nogo-A mRNA is present in sensory neurons of dorsal root ganglia and motoneurons in the lumbar spinal cord of the E10.5 mouse embryo. Expression of Nogo-A increases until E15.5. ROD, relative optical density. Scale bars: 100 μm, E10.5-E12.5; 200 μm, E15.5. (B) Whole-mount anti-neurofilament 160 immunostaining of wild-type and Nogo-A KO forelimbs at E12.5 and E13.5. At E12.5, thickness of the ulnar nerve was slightly increased in Nogo-A KO embryos (open arrows, upper panel), whereas its branching was decreased (open arrowhead, upper panel). Branching of the median nerve arbour was markedly reduced at both E12.5 and E13.5 in Nogo-A KO embryos (arrows, upper and middle panels). At E13.5, a reduction in the branching pattern of sensory cutaneous nerves was apparent; both major and fine branches were missing (arrow, lower panel). u.n., ulnar nerve; m.n., median nerve; s.c.n., sensory cutaneous nerves. Scale bar: 150 μm. (C) Innervation of wild-type and Nogo-A KO hindlimbs at E12.5 and E13.5. The width of the sciatic nerve and of its deep peroneal branch were increased in the absence of Nogo-A (open arrow and arrowhead, respectively; upper panel), whereas branching of the superficial peroneal nerve was reduced (arrow; upper panel). At E13.5, the tibial nerve divides into two major nerve trunks in wild-type embryos (arrows, lower panel). This bifurcation of the tibial nerve was disrupted in the absence of Nogo-A (arrow; lower panel) and its thickness was increased (open arrows, lower panel). s.n., sciatic nerve; t.n., tibial nerve. Scale bar: 150 μm. (D) Quantitation of the length and width of forelimb innervating nerves in wild-type and Nogo-A KO E13.5 embryos. UNW, ulnar nerve width; RNW, radial nerve width; MAW, median nerve arbour width; MAL, median nerve arbour length. Values represent means ± s.e.m. of 12 embryos per condition; *P<0.05, Student's t-test. (E) Quantitation of the length and width of hindlimb innervating nerves in wild-type and Nogo-A KO E13.5 embryos. TNW, tibial nerve width; TAL, tibial nerve arbour length; TAW, tibial nerve arbour width; SNW, sciatic nerve width; SPNL, superficial peroneal nerve length; DPNL, deep peroneal nerve length; DPNW, deep peroneal nerve width. Values represent means ± s.e.m. of 12 embryos per condition; *P<0.05 and **P<0.01, Student's t-test.