Fig. 3. Core planar polarity genes regulate the border cell actin cytoskeleton. Polar follicle cells are marked with white asterisks and border cells are marked with red dots. (A-G) Migrating border cell clusters, fixed to enhance preservation of the actin structures (see Materials and methods). In wild-type clusters (A) large actin-rich protrusions can be seen (arrowheads). In fz¹⁵/fz²³ (B), dsh¹ (C), stbm⁶ (D) and slbo-GAL4/UAS-fz- RNAi (E) mutants, the cytoskeleton appears fuzzy and large protrusions are rarely seen. Overexpression of fz (F) and stbm (G) under the control of slbo-GAL4 also disrupts the formation of large actin-rich protrusions. GAL4 experiments were carried out at 29°C. (H-L') Migrating border cell clusters stained for actin (red/white), expressing slbo-Gal4, UAS-GFP (green) at 25°C. Expression of dominant-negative Rho^N19 (H), Rho^N19 and fz- RNAi (I), fz-RNAi (J), dominant-active Rho^V14 (K), and Rho^V14 and fz-RNAi (L). The UAS-fz-RNAi insertion used was chosen because it gives weaker phenotypes than the insertion used for other experiments (e.g. panels E and N), with some actin-rich protrusions still being visible (J). Expressing dominant-negative Rho^N19 results in border cells becoming long, thin and not migrating effectively (H), and co-expressing fz-RNAi has no effect on this phenotype (I). Cells expressing dominant-active Rho^V14 become very round with an even cytoskeleton (K), and co-expressing fz-RNAi (L) ameliorates this phenotype, with the cells appearing less round and producing actin-rich protrusions (arrowheads). (M-N') Migrating border cell clusters, stained for actin (red), expressing GFP-RhoA (green/white). In wild-type clusters, GFP-RhoA colocalises with actin-rich protrusions at the cell surface (M), which are lost in cells expressing fz-RNAi under the control of slbo-GAL4 at 25°C (N), resulting in a partial redistribution of GFP-RhoA to the cytoplasm. Border cell clusters expressing fz-RNAi under the control of slbo-GAL4 showed an average cytoplasmic level of GFP-RhoA of 24.0% of peak membrane levels (n=10), compared with 15.4% for control clusters lacking the slbo-GAL4 driver (n=9), these results being statistically significant at the P<10^-5 level (t-test).