A semi-dominant mutation in the general splicing factor SF3a66 causes anterior-posterior axis reversal in one-cell stage C. elegans embryos.

Establishment of anterior-posterior polarity in one-cell stage Caenorhabditis elegans embryos depends in part on astral microtubules. As the zygote enters mitosis, these microtubules promote the establishment of a posterior pole by binding to and protecting a cytoplasmic pool of the posterior polarity protein PAR-2 from phosphorylation by the cortically localized anterior polarity protein PKC-3. Prior to activation of the sperm aster, the oocyte Meiosis I and II spindles assemble and function, usually at the future anterior pole, but these meiotic spindle microtubules fail to establish posterior polarity through PAR-2. Here we show that a semi-dominant mutation in the general splicing factor SF3a66 can lead to a reversed axis of AP polarity that depends on PAR-2 and possibly on close proximity of oocyte meiotic spindles with the cell cortex. One possible explanation is that reduced levels of PKC-3, due to a general splicing defect, can result in axis reversal due to a failure to prevent oocyte meiotic spindle microtubules from interfering with AP axis formation

Reversed polarity of P0 asymmetric cell division in <i>or430</i>ts zygotes.

<p>Time-lapse DIC images of wild type (A) and <i>or430</i>ts mutants (B–D), showing <i>or430</i>ts embryos with reversed (B), symmetric (C) and normal (D) cell division. Arrows indicate polar bodies. (E–G) Time-lapse confocal images of P₀ mitotic spindle orientation in wild-type and <i>or430</i>ts zygotes expressing a GFP fusion to ß-tubulin (and a GFP fusion to PIE-1 in F that labels P granules and cytoplasmic PIE-1)). Vertical lines in each image indicate 50% egg length. (H) P₀ mitotic spindle orientations in wild-type and <i>or430</i>ts zygotes (each n = 22). (I) P₀ cleavage furrow position along AP axis in wild-type (n = 26) and <i>or430</i>ts zygotes (n = 30). (J) Pronuclear meeting site along AP axis in wild-type (n = 22) and <i>or430</i>ts zygotes (n = 22). (K) Maximum P₀ mitotic spindle length in wild-type and <i>or430</i>ts zygotes (n = 22). (L) Duration of P₀ mitosis from pronuclear meeting to cleavage furrow ingression in wild-type and <i>or430</i>ts zygotes (each n = 22). In this and subsequent figures, t = 0 corresponds to pronuclear meeting.</p

Identification of <i>repo-1(or430</i>ts<i>)</i> causal mutation in the SF3a66 <i>C. elegans</i> ortholog F11a10.2.

<p>(A) Schematic of <i>repo-1</i> map position on chromosome IV and partial amino acid sequences of predicted proteins in wild-type and <i>or430</i>ts F11a10.2/<i>repo-1</i>, and in fly, human and fission yeast orthologs. Location of <i>tm4961</i> deletion indicated; whole genome sequencing of <i>repo-1(or430</i>ts<i>)</i> revealed no sequence changes in the neighboring gene, <i>lex-1</i>, which also is disrupted by the <i>tm4961</i> deletion (data not shown). (B) Loss of polarity reversal after RNAi knockdown of F11a10.2 in <i>repo-1(or430</i>ts<i>)</i> mutants, and in <i>repo-1(tm4961)/repo-1(tm4961)</i> mutants, assessed by measuring P₀ cleavage furrow position. Note that the single embryo with an apparent reversal in <i>repo-1(tm4961)</i> mutants may represent an example of a posteriorly positioned polar body, which occurs at low frequency in wild-type embryos. WT and <i>or430</i>ts data are same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106484#pone-0106484-g001" target="_blank">Figure 1I</a>. L4440 refers to the empty vector used as a negative control for feeding RNAi.</p

PKC-3 opposes and oocyte meiotic spindles promote AP axis reversal in <i>repo-1(or430</i>ts<i>)</i> zygotes.

<p>(A) Time-lapse confocal images of wild-type and <i>or430</i>ts oocyte Meiosis I and II in zygotes expressing GFP and mCherry fusions to ß-tubulin and Histone2B; t = 0 at ovulation. (B) P₀ cleavage furrow position along AP after reducing <i>pkc-3</i> gene dosage. P = 0.006 for an independent t test to compare the difference of the furrow position for <i>or430</i> versus <i>or430; pkc-3(−/+)</i>. (C) P₀ cleavage furrow position along AP axis after RNAi knockdown of <i>lin-5</i> and <i>unc-116</i>. P = 0.285 for an independent t test to compare the difference of the furrow position for <i>or430</i> versus <i>or430; unc-116(RNAi); lin-5(RNAi)</i>. WT and <i>or430</i>ts data in (B) and (C) are same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106484#pone-0106484-g001" target="_blank">Figure 1I</a>.</p

AP axis reversal in <i>repo-1(or430</i>ts<i>)</i> zygotes.

<p>(A) Time-lapse confocal images of wild type and <i>or430</i>ts zygotes expressing GFP fusions to ß-tubulin and PAR-2. (B) Time-lapse confocal images of wild type and <i>or430</i>ts zygotes expressing GFP fusions to ß-tubulin and PIE-1. (C) Kymographs of pseudocleavage furrow movement in wild-type zygotes and its absence in <i>or430</i>ts zygotes. Time in seconds before pronuclear meeting for both wild-type and <i>or430</i>ts are shown in wild-type images. (D) Quantification of GFP: PAR-2 levels in wild-type (n = 10) and <i>or430</i>ts (n = 10) zygotes; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106484#s4" target="_blank">Materials and Methods</a> for details. (E) Location along AP axis of P0 cytokinesis furrow ingression in wild-type and mutant zygotes. Arrows indicate polar bodies. P<0.001 for an independent t test to compare the difference of the furrow position for <i>or430</i> versus <i>or430; par-2(RNAi)</i>. Data for WT and <i>or430</i>ts are same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106484#pone-0106484-g001" target="_blank">Figure 1I</a>.</p