Visualization of the GAL4 expression pattern during oogenesis driving the RNAi knockdown, images from embryos expressing two alternative RNAi constructs, and evidence for the delocalization of Klp3A at the spindle midzone when Feo is knocked down. (A) V32–Gal4 drives expression during late oogenesis. Related to Fig. 2. A construct expressing V32–Gal4 driving UASp–GFP expression specifically in the female germline indicates that the peak expression of GFP is achieved only at late stages of oogenesis. It illustrates the expression pattern of UASp constructs under the same Gal4 driver, including the various RNAi constructs described here with maximum effect in late oogenesis. Scale bar, 10 µm. (B) Knockdown of Klp3A (40944) or Klp61F (33685) by RNAi leads to defective nuclear delivery to the embryo cortex. Maximum-intensity projections from 3D time-lapse videos of embryos inhibited for Klp3A or Klp61F expression by RNAi while expressing Jupiter::GFP (green) marking microtubules and H2Av::RFP (magenta) marking chromatin. These two alternative RNAi-expressing fly lines exhibited similar irregularity in nuclear distribution during the first interphase occurring at the cortex. Scale bar, 50 µm. (C and D) Depletion of Feo abolishes recruitment of Klp3A to the spindle midzone. (C) Snapshots from a time lapse of embryos expressing H2Av::RFP (magenta, left) and Klp3A::GFP (green, middle) during anaphase B or telophase. Feo-knockdown embryos failed to recruit Klp3A at the spindle midzone when compared with the control embryos expressing no feo RNAi. (D) Quantification of Klp3A::GFP intensity measured at the spindle midzone along the spindle axis in control and Feo RNAi (35467) embryos. Scale bar, 50 µm.