pico regulates Drosophila border cell migration via SCAR. (A–D) Analysis of time-lapse images of LifeAct-GFP–labeled border cells, using c306-GAL4 to drive the indicated genotypes (graphs indicate mean ± SEM; WT, n = 9; PVR^DN, n = 9; pico RNAi, n = 12; pico OE, n = 12; SCAR RNAi, n = 8; SCAR RNAi, pico OE, n = 7). (A) Graph summarizing migration rate/frame calculated using a custom macro (Poukkula et al., 2011). (B) Graph showing percentage frames from the first half of migration with tumbling border cell clusters (see Materials and methods). (B, right) GFP-labeled clusters display a polarized or tumbling phenotype. Bar, 15 µm. (A and B) Tests used were one-way ANOVA (P < 0.0001) and Tukey’s test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Graph showing number of cellular extensions per frame, irrespective of their direction. One-way ANOVA and Dunnett’s test were used. *, P < 0.05; **, P < 0.01. (D) Graph summarizing percentage extensions/frame at front, back, or sides of the cluster. (D, right) Image of a border cell cluster before and after image segmentation. The body of the cluster is shown in blue, cellular extensions at the front in green, the back in red, and the sides in white. White lines indicate quadrants, representing front, back, and sides for quantification. Bar, 5 µm.