Fig. 6. NMJ bouton phenotype of vps35. (A) Projections of confocal sections of wild-type (WT) and vps35 NMJs stained for the presynaptic marker Eps15 (green) and the largely postsynaptic marker Dlg (magenta). Insets show higher magnification of boxed areas. Arrowheads indicate examples of satellite boutons in vps35 NMJs, that are rare in WT; note that Eps15 labelling in these is completely surrounded by Dlg. (B) vps35 mutant larvae have approximately twice as many boutons as WT. The defect is partially rescued when Vps35 expression is driven by elav-Gal4 or BG57-Gal4 and completely rescued using a combination of these drivers. n=5-12 NMJs from 5-10 larvae; mean ± s.e.m. (C) Single confocal sections of NMJ boutons showing upregulation of pMad in vps35 mutant NMJs. Note the pMad puncta (green) predominantly at the inner surface of the mainly postsynaptic Dlg staining (magenta). (D) Quantification of pMad levels at the NMJ (n=10 boutons from five animals; mean ± s.e.m.). (E) The supernumerary bouton phenotype of vps35 larvae is suppressed by removal of a single copy of the BMP signalling components wit or Mad (wit/+, Mad/+ respectively) and more strongly by complete loss of Mad (Mad). n=5-11 NMJs from 5-9 larvae; statistical comparisons are with vps35. NMJs from muscles 6 and 7 in abdominal segment A3 were used for all analyses; mean ± s.e.m.