PHR controls growth cone size and filopodia length. a–e F-actin (green) and microtubules (magenta) were simultaneously labeled to visualize the growth cone cytoskeleton of regrowing Mauthner axons, in double transgenic Tg(hspGFF62a) Tg(UAS:lifeact-GFP-v2a-EB3-RFP) nonmutant siblings (a) or phr mutants (b) with long F-actin-rich filopodia labeled by yellow arrowheads (b); debris of the distal axon stump marked by white asterisks (a). Laser-mediated axon transection were performed in 5-day-old larvae and regenerating axons were imaged between 12 and 15 hpt. Scale bar in (b) is 10 µm. Schematic drawing of the phr mutant growth cone, showing how the growth cone was defined and how filopodia length was measured (c). The beginning of the growth cone was defined as an increase in the axon diameter of 25% or more compared to the proximal axon shaft, which usually correlated with an obvious increase in F-actin labeling. Filopodia were measured by a straight line from filopodia base to the end of the visible F-actin signal. Growth cone size as the area above a defined intensity threshold is displayed in (d). Growth cones were significantly larger in regrowing phr mutant axons compared to nonmutant siblings. P-value was determined using two-tailed Student’s t-test. Filopodia length was determined and the percentage of filopodia below or ≥ 5 µm shown as bar graphs (e). Significantly more filopodia ≥5 µm were seen in regrowing phr mutant axons compared to nonmutant siblings. P-value was determined using the Fisher exact test. N = 14 growth cones in 5 non-mutant siblings and n = 11 growth cones in 5 phr mutants larvae were measured; n = 94 filopodia in 5 nonmutant siblings and n = 119 filopodia in 5 phr mutant larvae were measured. More growth cones and individual channels are shown in Supplementary Fig. 3
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