Figure 4.

Wallerian degeneration in wild-type and milton MN axons. (A) Anatomical schematic of the larvae segmental nerve crush injury model (early third-instar larvae, 4 h post-crush). Pinching the ventral cuticle with forceps crushed the segmental nerves within a third-instar larva. The crush site was outlined by a break or reduction in horseradish peroxidase (HRP) labeling in the segmental nerves containing mCD8-GFP (C155-Gal4)-expressing axons from MN MARCM clones. Proximal and distal MN axons were readily traceable following axotomy, providing a tractable method of assessing single axon morphology and degeneration in vivo. (B–F) Montage of wild-type proximal, distal and far distal (300–500 μm from crush) axons at 4, 8, 16 and 24 h post-axotomy. The numbered panels display images of corresponding boxed areas in (B). Distal axon continuity was maintained at 4 h post-crush (B) with fragmentation of mCD8-GFP signal beginning 8 h post-crush (C and D, arrowheads demarcate mCD8-GFP axon fragments). By 16 h, all axons observed were fragmented (E) with a significant reduction in mCD8-GFP signal at 24 h (F). (G–J) Montage of milton^33-853 proximal, distal and far distal axons at 4, 8, 16 and 24 h post-axotomy. The numbered panels display images of corresponding boxed areas in (G). Wallerian degeneration followed a time course similar to wild-type MNs, with axon fragmentation beginning 8 h post-crush following a 4 h period of latency and completed by 16 h post-crush (G–H). (K) Morphological quantification of Wallerian degeneration in (B–J). Axon fragmentation was mildly delayed by milton^33-853, with a smaller fraction of fragmented axons observed 8 h post-axotomy, though all milton^33-853 axons were fragmented by 16 h as observed in wild-type controls. n = total number of MNs quantified per timepoint for each genotype. **P < 0.01, Fisher's exact test. Scale bars, 100 μm in (A), 50 μm for (B) and (G) and 20 μm in B3 and G3 for B1–F3 and G1–J3.