Figure 1. Development of abnormal cluster distribution and axonal branching in MyoD-/- mice. Confocal images of motor axons and AChR clusters in diaphragm muscles from BALB/c and MyoD-/- mice at P1, 14, 28, and 42 (top panels), and E14.5 and E15.5 (bottom panels). Muscles were reacted with a mixture of antibodies against neurofilament and synaptophysin and with an FITC-conjugated secondary antibody to label preterminal and terminal portions of the motor axons (A–F, a–f) and with rhodamine-conjugated α-BuTx to label AChRs (A′–F′,a′–f′). In the BALB/c diaphragms, at all postnatal ages, neuronal branches (A–D) and AChR clusters (A′–D′) are confined to the middle of the muscle (in a discrete endplate band), whereas in the diaphragms of MyoD-/- mice, there is extensive ramification of neuronal branches (a–d) throughout the muscle, extending to the edge of the diaphragm (a, d, arrowheads). AChR clusters are distributed throughout the muscle, corresponding to the distribution of the axon terminals (a′–d′). At E14.5 a discrete band of AChR clusters is found in the middle of the diaphragms of both BALB/c (E′) and MyoD-/- mice (e′). The FITC and rhodamine images were overlapped (E, F, e, f), showing that some terminal axons are not apposed to AChR clusters at E14.5 (E, e, arrows.). By E15.5 motor axons have grown out of the central zone of the muscle fibers, and new AChR clusters are formed at the axon terminals of the MyoD-/- mice (f), resulting in a wider, more diffuse endplate band (f′) than is found in age-matched BALB/c muscle (F′). Scale bars, 100 μm. For each age group, micrographs are at the same magnification.