Neuronal expression of stathmin RNAi reduces NMJ size and results in axon retraction. A, Images of single optical sections of motor nerves immunostained with α-Stathmin (green) and α-HRP (red). Immunostaining of WT nerves reveals Stathmin staining in both the outer perineurial glial cells that surround the HRP-positive region and within the HRP-positive region itself, which contains axons and inner ensheathing glial cells. In contrast, Stathmin signal is significantly decreased in the stathmin mutant (stai^B200/stai ^B200). Neuronal expression of stathmin RNAi (UAS-stai RNAi/+; +/+; GeneSwitchELAV/+) reduces Stathmin signal in the HRP-positive region of the axons. Conversely, glial expression of stathmin RNAi (UAS-stai RNAi/+; +/+; repo-Gal4/+) decreases Stathmin signal in the glial cells and allows clearer visualization of axonal Stathmin. B, Images of segment 4 NMJs from WT (GeneSwitchELAV/+) and following neuronal expression of stathmin RNAi (UAS-stai RNAi/+; +/+; GeneSwitchELAV/+). Immunostaining for α-DVGLUT (green) and α-Dlg (red) reveals a reduction in muscle 4 NMJ size and bouton number following neuronal expression of stathmin RNAi (middle row) compared to WT (top row). Axon retractions are also detected at muscle 6/7 NMJs following stathmin RNAi expression (bottom row) by the presence of postsynaptic Dlg footprints (arrowhead). Scale bars: 10 μm.