Figure 3

Uncoupling muscle-dependent from Met-dependent axonal branching. (A) Anti-neurofilament (NF) immunohistochemistry on E12.5 and E13.5 hindlimbs of WT and met mutants. (B) MyoD ISH showing differentiating muscles in E13.5 and E14.5 hindlimbs. (C) Recapitulative schemes positioning hindlimb muscles and nerves with respect to each other. Colour code for nerves; green: ventral nerves not in the focal plane and unaffected by lack of Met signalling; red: peroneal nerve motor branches, innervating the dorsal limb compartment, corresponding to the common peroneal nerve as it emerges from the sciatic plexus. The cutaneous sensory peroneal nerve branch is shown in blue. (D) Quantification parameters. α: angle between the transverse and distal portion of the deep peroneal nerve. β: angle between common and deep peroneal nerve portions, used as internal morphological landmark. Blue arrows: contact sites with ta and edl muscles where the number of peroneal nerve side branches were quantified. (E) The nerve phenotype severity was assessment by measuring the α and β angles, in WT (n=12), met^d/d (n=10), met^2P/2P (n=7), and met^2S/2S (n=9) E12.5 and E13.5 hindlimbs. Each dot represents one embryo side. The same colours representing the genotypes were used in all graphs. (F) Plot showing the respective numbers of side-branches per peroneal nerve (pooling ta and edl branches), in E13.5 WT (n=6), met^d/d (n=7), met^2P/2P (n=6), and met^2S/2S (n=8) hindlimbs. Differences between WT and met^d/d embryos, and between met^2P/2P and met^2S/2S embryos, respectively, are significant (Mann-Whitney test, p<0.01).) (G) Measurements of ta and edl muscle surface were performed on WT (n=6), met^d/d (n=6), met^2P/2P (n=8), and met^2S/2S (n=7) E13.5 and E14.5 hindlimbs stained with MyoD ISH. Areas were expressed as percentage of the mean area in WT embyos (represented as 100%). The plot therefore shows only met^2P/2P and met^2S/2S values, each dot representing one embryo side.