The responsiveness of myotubes in E14.5 diaphragms to agrin is restricted to their central regions. A, A′, E14.5 diaphragms cultured for 18 h in the absence (A) and presence (A′) of 10 nm chicken mini-agrin cN25₇C21B₈. Note that, despite the presence of agrin, clusters are confined to the central region of the myotubes. Scale bar, 40 μm. B, B′, Diaphragms of MCK–cmag_B8 mice expressing mini-agrin from myotubes in vivo at E13.5. B, After staining for agrin immunoreactivity; B′, same region stained for AChR clusters. Note absence of AChR clusters in lateral regions (*) of the diaphragm despite uniform agrin transgene expression along entire myotubes. C, C′, Same as in B but from w.t. littermate. Scale bar, 25 μm.