Figure 3. Different filopodial signatures accompany separate circuit formation steps.
Slow (30 min interval) time-lapse imaging of pupal brains dissected at P + 20% (a), P + 40% (b) and P + 55% (c) in comparison with in vivo fixed controls at the same stages. The same growth cones were analyzed for all live imaging experiments while different samples from parallel aged pupae had to be dissected for the in vivo controls. All photoreceptors were labeled with myr-mRFP and R7 cells were sparsely labeled with CD4-tdGFP using GMR-FLP through MARCM. (a) As the R7 and R8 layers go through their initial separation (upper panel), R7 terminals have numerous filopodia that invade neighboring columns (lower panel), which are pruned around P + 40% both ex vivo and in vivo. (b) As the layers start to reach their final configuration, R7 terminals form a bipartite structure around P + 50%. Filopodia numbers remain low. Around P + 55%, more (shorter) filopodia are observed again as R7 axon assumes a brush-like look. (c) After P + 55% shorter filopodia are pruned and R7 growth cones form new, longer filopodia that are fewer in number and have bulbous tips (arrows). Quantifications of (d) total number of filopodia per growth cone and e, mean length of filopodia through the ex vivo experiments (a-c) and respective in vivo controls. Error bars depict SEM. Scale bars, 5 μm.
Figure 3—figure supplement 1. Filopodial dynamics are restricted to the growth cone and axon shaft inside the medulla neuropil.
