<i>vha-19(RNAi)</i> adults produce fewer oocytes beyond 24 hours of exposure to <i>vha-19</i> dsRNA.

<p>A) The average number of eggs laid every 24 hours by <i>control(RNAi)</i> or <i>vha-19(RNAi)</i> adults feeding on dsRNA. Error bars represent the standard error of the mean (s.e.m). ***indicates p<0.001. B) The average number of oocytes in the proximal oviduct of either wild type or AZ212 (pie-1::H2B::GFP) <i>C. elegans</i> fed on control or <i>vha-19</i> dsRNA for 24 or 48 hours. C) The proximal oviduct of AZ212 <i>C. elegans</i> fed control or <i>vha-19</i> dsRNA for 24 hours. White arrows indicate normal oocyte nuclei. Endomitotic oocytes in the oviduct are indicated by a square bracket. Note that both oocytes with multiple nuclei (most proximal oocyte in square bracket), and oocytes in which the chromosomes have started to decondense prematurely (indicated by the green background of the two most distal oocytes in the square bracket), are endomitotic. The irregular, bright, particulate dots in the distal gonad (dg) are autofluorescent gut granules. Scale bars indicate 10 µm. D) The percentage of endomitotic oocytes observed in the oviducts of wild type or AZ212 <i>C. elegans</i> fed control or <i>vha-19</i> dsRNA from the L4 stage for 24 or 48 hours.</p

The progeny produced by mating <i>fog-2</i> male <i>C. elegans</i> with <i>fog-2</i> female <i>C. elegans</i> fed on either control or <i>vha-19</i> dsRNA.

<p>∧Significantly different to average brood size (p<0.01) or number of progeny that reached adulthood (p<0.0001) for <i>fog-2(q71) V</i>; <i>control(RNAi)</i>l x <i>fog-2(q71) V</i>; <i>control(RNAi)</i> mating.</p>*<p>Not significantly different to average brood size/number of progeny that reached adulthood for <i>fog-2(q71) V</i>; <i>vha-19(RNAi)</i> x <i>fog-2(q71) V</i>; <i>vha-19(RNAi)</i> mating (p>0.05).</p

<i>vha-19(RNAi)</i>embryos are osmotically sensitive and appear to have a cytokinesis defect.

<p>A) Embryos cut out of the uterus of wild type (A-B,F) or pie-1::H2B::GFP [AZ212] (E) adult <i>C. elegans</i> fed control or <i>vha-19</i> dsRNA for 24 hours. Note the presence of an eggshell in all embryos in A (arrow heads), despite the random clusters of nuclei and compression of some <i>vha-19(RNAi)</i> embryos. Note also the presence of a polar body (pb) in a <i>vha-19(RNAi)</i> embryo in E, even though the nuclei are not cellularized (inset, bright field image), and that in F, non-cellularized, small clusters of nuclei (square bracket) are surrounded by cleavage furrows (arrows) in the <i>vha-19(RNAi)</i> embryo. C) The percentage of <i>control(RNAi)</i> and <i>vha-19(RNAi)</i> embryos that swelled or shrunk in deionized water (mQ H2O), 150 mM potassium chloride (KCl) and 300 mM KCl. Examples of embryos from this osmolarity experiment are shown in B. D) Embryos in the uterus of adults fed control or <i>vha-19</i> dsRNA for 48 hours and stained with Acridine Orange. Note that embryos in the uterus of <i>control(RNAi)</i> adult excluded Acridine Orange (arrows) whereas the embryos in the uterus of the <i>vha-19(RNAi)</i> adult were permeable to Acridine Orange (bracket). Scale bars represent 100 µm in D and 10 µm in A-B,E and F.</p

Loss of <i>lig-4</i> prevents chromosomal fusion in <i>com-1</i> mutants, but does not restore viability.

<p>(A) Two representative pictures of diakinesis nuclei of animals of the indicated genotype. White arrows point out chromosomal fragments (B) Percentage progeny survival; values are the average of 3 independent experiments, error bars represent S.E.M. (C) Percentage of diakinesis nuclei that show chromosomal fragments; n = number of germlines analyzed. Scale bars, 5 µm. *The difference between these genotypes was highly significant (p<0.001 by Fisher's exact test, two tailed).</p

EXO-1 is required for meiotic recombination in absence of COM-1.

<p>(A) Representative image of diplotene nuclei of <i>com-1 cku-80 exo-1</i> triple mutant animals that express a ZHP-3::GFP transgene (left: GFP signal only, right: merge of GFP and DAPI signal) (B) Representative picture of a diakinesis nucleus in <i>com-1 cku-80 exo-1</i> triple mutants germlines (C) RAD-51 immunostaining of mid-pachytene nuclei (zone 5) in <i>com-1 cku-80 exo-1</i> mutant germlines; merge of RAD-51 (red) and DAPI signal (blue) (D) Percentage progeny survival of animals of the indicated genotype; values are the average of 3 independent experiments*, error bars represent S.E.M. (E) Frequency distribution of DAPI-stained entities at diakinesis*. n = number of germlines analyzed. The <i>com-1 cku-80 exo-1</i> triple mutants occasionally showed >12 DAPI bodies due to chromosomal fragmentation. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003276#pgen-1003276-g007" target="_blank">Figure 7E</a> for quantification. Scale bars, 5 µm. *These experiments were performed in parallel to those depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003276#pgen-1003276-g001" target="_blank">Figure 1B and 1D</a>; reference values are depicted again here.</p

LIN-61 is dispensable for DNA damage checkpoints in the germline.

<p>(A) Schematic diagram of the hermaphrodite germline. Cell cycle arrest (as in B–C) occurs in the mitotic zone and apoptosis (D–E) occurs at the bend of the germline. DTC, distal tip cell; TZ, transition zone. (B) Maximum projections of DAPI-stained mitotic nuclei 24 hours after irradiation with 60 Gy or mock-treatment. (C) Quantification of mitotic cell cycle arrest, error bars are s.d. (D) DIC images of pachytene stage nuclei 24 hours after irradiation with 60 Gy or mock-treatment. Arrowheads mark apoptotic corpses. (E) Quantification of apoptotic corpses per germline arm. Error bars represent s.d. (F) Quantification of <i>egl-1</i> mRNA by qRT-PCR, normalised to untreated wild types. Total RNA was isolated from mixed populations of developmentally staged young adults 24 hours after irradiation with 120 Gy, or mock treatment.</p

Model for meiotic recombination in <i>C. elegans</i>.

<p>In wild-type germlines, MRE-11 may create substrates at meiotic DSBs that allow COM-1 to efficiently remove Ku (and SPO-11). When COM-1 function is perturbed, MRE-11 mediated processing may still release SPO-11 bound oligos. However, MRE-11 activity alone is not sufficient to counteract Ku binding and prevent toxic NHEJ activity. Without COM-1 and Ku, SPO-11 is removed and EXO-1 promotes DNA end resection and allows the obligate COs to be formed. See text for further details.</p

VHA-19 is required for uptake of vitellogenin via trafficking of the RME-2 receptor.

<p>A) VIT-2::GFP <i>C. elegans</i> [DH1033] were fed control or <i>vha-19</i> dsRNA from the L4 stage for 48 hours and the oocytes examined. VIT-2::GFP did not reach the oocytes in adults fed <i>vha-19</i> dsRNA, instead it accumulated between the oocytes (thin arrows). Fat arrows indicate agglomerates of VIT-2::GFP. B) RME-2::GFP was not trafficked to the plasma membrane in RME-2::GFP; <i>vha-19(RNAi)</i> adults (arrows and dotted circle), whereas RME-2::GFP did reach the plasma membrane in worms fed control dsRNA (thin arrows between oocytes). Worms were fed <i>vha-19</i> or control dsRNA for 48 hours. Arrowheads in bright field panels indicate individual oocytes. Scale bars represent 10 µm.</p

<i>C. elegans</i> arrest as larvae when fed on <i>vha-19</i> dsRNA from the first larval stage.

<p><i>C. elegans</i> arrest as larvae when fed on <i>vha-19</i> dsRNA from the first larval stage.</p

<i>vha-19(RNAi)</i> adults lay compressed eggs despite the presence of sperm in the spermatheca.

<p>Wild type (A-B, D) or <i>rrf-1</i> (C) <i>C. elegans</i> were fed control or <i>vha-19</i> dsRNA from the L4 stage for 16–48 hours. A) The proximal gonad of a <i>control(RNAi)</i> and <i>vha-19(RNAi)</i> adult after feeding on dsRNA for 16 hours. B) The percentage of <i>control(RNAi)</i> or <i>vha-19(RNAi)</i> adults that had one or more compressed (squashed) eggs present in their uterus from 16–48 h. At least 89 worms were scored for each experimental group at each time point. C) The proximal gonads of <i>rrf-1(pk1471) C. elegans</i> after feeding on dsRNA for 24 hours. D) A typical proximal gonad of a wild type <i>C. elegans</i> that was fed <i>vha-19</i> dsRNA from the L4 stage for 24 hours. Note the presence of newly released, compressed eggs in the uterus (bracket), even though there are sperm in the spermatheca (enclosed by a white box and magnified in E. Examples of sperm are indicated by arrows or enclosed by a white circle). In all images black arrowheads indicate normal embryos, and a bracket indicates compressed eggs. The oocytes (oo) and spermatheca (sp) are also indicated. v =  vulva. Scale bars represent 100 µm in A and C and 10 µm in D.</p