FIG. 4.

Innervation of the diaphragm by the phrenic nerve. (A) In control mice at E14.5, the axons of the phrenic nerve, stained green with antibodies against neurofilament and synaptophysin, have reached the diaphragm, branched, and extended both dorsally and ventrally to the full extent of the muscle. (B) In 9ThW/1Acrg littermate mice at E14.5, the phrenic nerve is less robust and fails to reach large portions of the diaphragm. In particular, the ventral portion of the muscle is not innervated. (C and D) Higher-magnification views of the regions indicated in panels A and B shows that the aneural muscle in 1Acrg/9ThW mice has a pattern of postsynaptic differentiation that is consistent with muscle that has never beeninnervated. (E) By E18.5, control mice have a similar but more elaborate pattern of motor innervation, with the nerves terminating on plaques of AChRs, stained red with rhodamine-conjugated α-bungarotoxin, on the muscle fibers. (F) In 9ThW/1Acrg mice at E18.5, the ventral diaphragm is still not innervated and the pattern of aneural muscle is similar to that at earlier ages. The defects in innervation of the diaphragm correspond to deficiency combinations that cause lethality due to respiratory distress. (G) Mice homozygous for 15DttMb show defects in the phrenic nerve that are very similar to those seen in 9ThW/1Acrg mice. (H) However, 48UThc/1Acrg mice are viable postnatally and show no defects in the diaphragm at E18.5. (I and J) Higher-magnification views of the 15DttMb diaphragm and a control diaphragm in the ventral affected region. In all cases, the right side of the diaphragm is shown, dorsal is left, ventral is right, Mid indicates the ventral midline, and the arrowheads indicate regions of incomplete innervation. Bars, 1 mm (A and B) and 1.6 mm (E to H).