Fig. 4. Lrp4 is required for the initiation of AChR clustering. E13.5 (A-F) and E14.5 (G-L) wild-type (A-C,G-I) and mutant (D-F,J-L) diaphragms, with neuronal projections visualized with neurofilament (A-F, green) or HB9-GFP (G-L, green) and AChRs marked by -bungarotoxin (red). Before innervation, pre-clustering of AChRs does not occur in Lrp4 mutants (compare B to E). At E14.5, mutant phrenic nerves show longer primary branches that do not stop in the endplate region. AChR clusters are still undetectable in Lrp4 mutants at this stage of NMJ initiation (K). (M-P) Wild type (M,O) and Lrp4 mutant (N,P) cultured myotubes, either untreated (M,N) or following overnight treatment with recombinant Agrin (O,P). AChR clusters (visualized with -bungarotoxin in red) form in myotubes derived from wild type but not Lrp4 mutants.