Spermatid cysts elongate in the wrong direction in <i>apkc</i> or <i>orb2</i> mutants.

<p>A) Frequency of testes that have at least one spermatid cyst polarized in the wrong orientation so that clustered spermatid cyst nuclei are found in the apical region instead of towards the base of the testis. <i>apkc⁴⁸</i> (<i>apkc^ex48</i>), <i>06403</i> (<i>apkc⁰⁶⁴⁰³</i>) and <i>apkc⁵⁵</i> (<i>apkc^ex55</i>) are <i>apkc</i> mutant alleles. <i>34332</i> and <i>35140</i> are UAS-<i>apkc</i> RNAi lines (dsRNA targeting sites marked in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004380#pgen-1004380-g003" target="_blank">Fig. 3A</a>), while the Gal4 drivers are <i>nos</i> or MTD. MTD has three GAL4 drivers, <i>pCOG-Gal4</i> which has an <i>otu</i> promoter, plus two different <i>nos</i> drivers, <i>NGT-40</i> and <i>nanos</i>-<i>Gal4</i>. Error bars are standard deviation based on three experiments. Total number of testes scored for each genotype are (from left to right): Fig. A1: <i>apkc^ex48</i>: 36; <i>apkc⁰⁶⁴⁰³/+</i>:33; <i>apkc^ex55</i>:27; MTD/UAS-<i>apkc:34332</i>:43; <i>nos-GAL4/</i>UAS-<i>apkc:35140</i>: 18; MTD/UAS-<i>apkc:35140</i>:16; <i>orb³⁶/+</i>:55; <i>orb³⁶</i>: 50; <i>orb2^ΔQ</i>: 86; WT: 65. Fig. A2: <i>apkc⁰⁶⁴⁰³/+</i>: 38; <i>apkc⁰⁶⁴⁰³/</i>+; <i>orb2^ΔQ</i>/+:40; <i>apkc⁰⁶⁴⁰³/</i>+; <i>orb2³⁶</i>/+: 49; Fig. A3: <i>apkc^ex48/+</i>: 44; <i>apkc^ex48/</i>+; <i>orb2^ΔQ</i>/+: 39; <i>apkc^ex48/</i>+; <i>orb2³⁶</i>/+: 39; <i>apkc^ex55/</i>+: 27; <i>apkc^ex55/</i>+; <i>orb2^ΔQ</i>/+: 28; <i>apkc^ex55/</i>+; <i>orb2³⁶</i>/+: 37. B-D) Whole mount antibody staining of wild type or <i>apkc</i> mutant testes. Blue, DNA; red, Orb2. B) Spermatid nuclei clusters (arrow) are found in the spermatogonial region of the testis in <i>apkc</i> hypomorphic alleles. The example shown here is <i>apkc^ex55</i>. D) In the hypomorphic alleles, most spermatid nuclei clusters are located as in wild type on the basal side of testes. D) In MTD/UAS-<i>apkc:35140</i> testes, multiple spermatid nuclei clusters are found in the apical region (arrows). (Not all spermatid cysts express Orb2 and/or are in focus). E) In wild type testes spermatid cyst nuclei are never seen at the apical end of the testis. F) aPKC (green) and Orb2 (red) in a misoriented elongating <i>apkc^ex55</i> spermatid cyst. Note that the Orb2 comet head is present. G) aPKC (green) and Orb2 (red) in a misoriented elongating MTD/UAS-<i>apkc:35140</i> testis. Note that the Orb2 comet is absent. This is also the case for correctly oriented MTD/UAS-<i>apkc:35140</i> spermatid cysts (not shown). All images are orientated with apical side of the testes to the left and basal to the right. Scale bars: 50 µm.