Dynein is required for posterior <i>oskar</i> mRNA localization.

<div><p>(A) Ovarian lysates were prepared from flies expressing shRNAs against <i>luciferase</i> (lane 1) and <i>dhc</i> (lanes 2 and 3). The lysates were run on a gel and analyzed by western blotting using the indicated antibodies. Dhc levels were dramatically reduced in <i>dhc</i> shRNA expressing lysates. By contrast, Khc and gamma-tubulin levels were unaffected. This experiment was repeated three times.</p> <p>(B-D) Ovaries were dissected from flies expressing shRNAs against <i>luciferase</i> (B) and <i>dhc</i> (C, D). The egg chambers were stained with an antibody against Dhc and were counterstained with TRITC-conjugated Phalloidin (actin stain). The panels on the left depict early-stage egg chambers (S5), whereas those on the right are mid-stage egg chambers (S10). Note that whereas Dhc levels were the same in early-stage egg chambers, the level of Dhc was greatly reduced in the germline of <i>dhc</i> shRNA expressing mid-stage egg chambers. The arrows indicate the strong signal for Dhc in the overlying somatic follicle cells. Dhc depletion was verified using immunofluorescence on more than ten occasions.</p> <p>(E-G) Ovaries were dissected from flies expressing shRNAs against <i>luciferase</i> (E) and <i>dhc</i> (F,G). The egg chambers were processed for in situ hybridization using anti-sense probes against <i>oskar</i> mRNA. The egg chambers were imaged under conditions of low-gain (E, F and G) and high-gain (E’, F’ and G’). Expression of shRNA targeting <i>luciferase</i> had no effect on the localization of <i>oskar</i> mRNA. By contrast, depletion of Dhc resulted in significant <i>oskar</i> mRNA delocalization. <i>oskar</i> mRNA was delocalized in 82% of egg chambers expressing <i>dhc</i> shRNA-A (n=191) and in 87% (n=106) of egg chambers expressing shRNA-B. (H-I) In order to validate the <i>oskar</i> mRNA in situ signal, control (H) and Dhc depleted (I) ovaries were dissected and processed for in situ hybridization using sense probes against <i>oskar</i> mRNA. Ten times more sense probe was used in this experiment in comparison to the anti-sense probes used in panels E-G. Despite the abundance of probe, no in situ signal was observed. </p> <p>(J-K) Egg chambers from flies expressing shRNAs targeting <i>luciferase</i> (J) and <i>dhc</i> (K) were stained with an antibody against Staufen. The egg chambers were also counterstained with Alexa 633-conjugated Phalloidin. Staufen, which serves as a marker for <i>oskar</i> mRNA, is localized at the posterior pole in control oocytes. In contrast, Dynein depletion results in greatly reduced posterior Staufen (73% affected; n=130).</p>