syd-1/liprin-α/wrd function in a linear pathway to prevent ectopic distribution of SV proteins at the distal axon. A, Representative confocal images of the distal axons of muscle 12/13 NMJs costained with anti-DVGlut and anti-HRP (not depicted, used as a reference for the distal axon) antibodies in third-instar larvae of wild-type, syd-1, liprin-α, or wrd mutants, as well as each of the above genotypes plus neuronal expression of each of three individual rescuing transgenes (D42–Gal4/UAS–GFP–Syd-1, D42–Gal4/UAS–liprin-α–GFP, D42–Gal4/UAS–GFP–Wrd; for full description of genotypes, see Materials and Methods). White brackets show terminal axons crossing muscle 13 to innervate muscle 12. B, Quantification of the distal axon vesicle clustering (measured by average intensity of DVGlut at muscle 12/13 terminal axons) in third-instar larvae of flies presented in A. n.s., Not significant; res, rescue. *p < 0.001. Error bars denote SEM.