Gastrocnemius muscle was dissected out in P0 pups and sectioned longitudinally, or, in some cases, the whole leg at the level of gastrocnemius muscle with tibial nerve was sectioned in cross section. Sections were incubated with monoclonal S46 antibody (gift of Dr. Stockdale) reactive to the spindle-specific slow-tonic myosin heavy chain isoform and developed by Vector mouse on mouse kit as described above, followed by rabbit anti-NF-M antibody (Chemicon) and CY3 conjugated goat anti-rabbit antibody (Chemicon) in a sequential double-labeling protocol. In another method, DiI crystals were placed in DRGs of E17 embryos from different genotypes (n = 2), and the gastrocnemius muscle was isolated and sectioned into 40-μm-thick slices on a vibratome.