Fig. 7.

Multiplexed PCR tagging reveals vamp1/2 co-expression in motor axons. (A) A larva in which endogenous Vamp1/2 proteins are both tagged shows that synaptic boutons in individual motor axons exhibit molecular heterogeneity and can be Vamp1⁺ (green arrowheads), Vamp2⁺ (magenta arrowheads) or both Vamp1/2⁺ (white arrowheads). (B) An individual motor axon in which Vamp1/2 proteins are labelled using a Gal4/UAS overexpression strategy. All synaptic boutons appear to be Vamp1/2⁺ (white arrowheads). (C) mEGFP and mCherry fluorescence intensity profiles along the indicated arrows in motor axon terminals shown in A,B. Arrowheads indicate the terminals highlighted in A,B. Note mEGFP and mCherry overlap in overexpressed but not endogenously labelled terminals. (D) To estimate the fluorescence signal along the axon outside the synaptic boutons, the lowest 10% of the fluorescence intensity value was averaged per axon profile (plotted in arbitrary units, AU). Note significant increase in overexpression strategy. Circles indicate individual axon profiles (five axons from three endogenously tagged larvae and five axons from five overexpressing larvae) and bars indicate the median. Error bars indicate the interquartile range. **P<0.01 (two-tailed Mann–Whitney test). (E) The proportion of Vamp1⁺, Vamp2⁺ and Vamp1/2⁺ boutons along each motor axon. The overexpression strategy masks neuromuscular junction molecular heterogeneity. Bars indicate the proportion of each type of bouton averaged from five axons each (from three endogenously tagged and five overexpressing larvae), and error bars indicate the 95% confidence intervals (Wilson/Brown calculation). ***P<0.0001 (Fisher's exact test). Scale bars: 5 µm.