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

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

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

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

Pupfish_contigs.fa

de-novo partial reference genome for Cyprinodon diabolis containing 793,068 RAD contigs ranging between 300 - 796 bp

cramfiles

Individual alignment files in CRAM format

BEAST_xml_files

XML files used in *BEAST analysi

Phylogenetic_data_sets

fasta formatted sequence files for 200 random RAD loc

<i>mei-1</i> mutants.

<p>A. DIC time-lapse images of wild-type, <i>mei-1(or642 </i>ts<i>) and mei-1(or646 </i>ts<i>)</i> embryos. In the <i>mei-1</i> mutants the polar bodies were large and misshapen and embryos contained multiple [top <i>mei-1(or642 </i>ts<i>)</i> embryo and <i>mei-1(or646 </i>ts<i>)</i>] or zero maternal pronuclei (second <i>mei-1(or642 </i>ts<i>)</i> embryo). The two <i>mei-1(or642 </i>ts<i>)</i> embryos were obtained from a hermaphrodite shifted to the restrictive temperature for 30 minutes, the <i>mei-1(or646 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 7 hours prior to imaging. White arrowheads indicates polar bodies, black arrowheads indicate multiple maternal pronuclei, the black arrow denotes multiple nuclei per cell at the two cell stage, and the “p” refers to the paternal pronucleus in an embryo lacking a maternal pronucleus. Times in min:sec are given relative to nuclear envelope breakdown (NEBD). Scale bar, 10 µm. B. Defect maps of individual embryos observed during time-lapse recordings: embryos are listed on the left and phenotypes are listed on the top: 1; normal polar body size, 2; normal pronuclear number, 3; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at the restrictive temperature for 30 minutes. C. Amino acid alteration in the mutants. Asterisk indicates the changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein.</p

<i>par-2</i> mutants.

<p>A. DIC time-lapse images of wild-type <i>par-2(or373 </i>ts<i>)</i>, <i>par-2(or539 </i>ts<i>)</i>, and <i>par-2(or640 </i>ts<i>)</i> embryos. The blastomeres in the <i>par-2</i> mutants were of similar size at the two cell stage and initiated mitosis simultaneously, in contrast to the wild type. The <i>par-2(or373 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 5 hours prior to imaging. The <i>par-2(or539 </i>ts<i>)</i> and par-<i>2(or540 </i>ts<i>)</i> embryos were obtained from hermaphrodites shifted to the restrictive temperature for 30 minutes prior to imaging. Arrows indicate mitotic spindles at the two cell stage. Times in min:sec are given relative to AB NEBD. Scale bar, 10 µm. B. Defect map for individual embryos observed during time-lapse recordings, embryos are listed on the left and phenotypes are listed on the top: 1; Normal one cell embryo; 2; assymetric two cell embyro, 3; asynchronous two cell divisions. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites transferred to the restrictive temperature for 30 minutes. C. Amino acid alteration in the <i>par-2(or373 </i>ts<i>)</i> mutant. Asterisk indicates the changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein.</p