hpo and wts are required in polar cells for the migration of border cell clusters and the induction of outer border cells. upd-Gal4 was used to overexpress or knock down genes specifically in polar cells. UAS-GFP or UAS-RFP driven by upd-Gal4 was used as a control (A and I). UAS-GFP was included in B–G. The flies were dissected after being grown at 29° for 5–6 days. The ovaries were immunostained with anti-Fas3 and anti-GFP (A–G) or anti-Slbo (I–N). Stage-10 egg chambers were selected and oriented as anterior toward the left (A–G and I–N). High magnification views of border cell clusters are shown on the right. (A) In the UAS-GFP control, a border cell cluster migrated normally and reached the oocyte-nurse-cell border. (B) Knockdown of hpo impaired border cell migration. (C and D) Knockdown of wts with two RNAi lines impaired border cell migration. Extra Fas3-positive cells were observed. (E) Knockdown of yki did not impair border cell migration. (F) Knockdown of sd did not impair border cell migration. (G) Overexpression of hpo did not impair border cell migration. (H) Quantification and percentage distribution of border cell migration. Wilcoxon rank-sum test, **P < 0.01. (I) In the UAS-RFP control, a border cell cluster with at least five Slbo-positive cells migrated normally and reached the oocyte-nurse-cell border. (J) No Slbo-positive cell was observed, and the border cell cluster did not migrate when wts was knocked down in polar cells. (K) No Slbo-positive cell was observed, and the border cell cluster did not migrate when yki-S111A.S168A.S250A (yki-3SA) was expressed in polar cells. (L) A border cell cluster with at least four Slbo-positive cells migrated normally when sd was knocked down in polar cells. (M) A border cell cluster with at least four Slbo-positive cells migrated normally and reached the oocyte-nurse-cell border when yki-3SA was expressed and sd was knocked down in polar cells. (N) A border cell cluster with at least four Slbo-positive cells migrated normally and reached the oocyte-nurse-cell border when wts was knocked down and upd was overexpressed in polar cells. (O) Quantification and percentage distribution of border cell migration. (P) Quantification and percentage distribution of Slbo-positive cell numbers in stage-10 egg chambers. Wilcoxon rank-sum test, **P < 0.01 comparing with control; ##P < 0.01. Bar, 20 μm in A–G and I–N.