Fig. 5. Increased nerve branching and broadening of the innervation band in ^-/- muscles. Whole-mount diaphragm muscles were immunostained with antibodies against neurofilament (A,B), syntaxin (C,D), synaptophysin (E,F) or synaptotagmin 2 (Syt2) (G,H). (A,B) The hemi-diaphragm; (C-H) the dorsal quadrant diaphragm. In E13 wild-type muscle, collateral axons emanated from the nerve trunk in an orderly fashion and were mainly confined to the central region of the muscle (arrowheads in A); in E13 ^-/- muscle, noticeable increases of axons were observed and distributed across a broad region of the muscle (B). Similarly, at E15.5, intramuscular nerves were confined to the central region of wild-type muscle (C,E,G), but projected aberrantly to a broad region of the ^-/- muscles (D,F,H). Numerous puncta labeled by either synaptophysin or Syt2 antibodies at the nerve terminals are observed in both the wild-type and ^-/- muscles (arrowheads in E-H, also see inset in G,H). Scale bar: 200 µm.