Inability to initiate recombination during triplo-X meiosis impairs establishment of exclusive, pairwise interactions.

<p><b>A.</b> Major classes of X chromosome synapsis configurations in triplo-X nuclei illustrated by 3-D surface renderings of individual nuclei stained for HIM-8 (white), HTP-3 (red), SYP-1 (green), and DAPI (blue). Scored nuclei were from the latter half of the region corresponding to mid-pachytene in diploids, which corresponds to the location of S-phase labeled nuclei at the 24 h time point (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen-1003963-g003" target="_blank">Figure 3B</a>). Red dotted traces indicate regions of HTP-3 staining where SYP-1 was not detected, and yellow dotted traces indicate regions of HTP-3 and SYP-1 overlap associated with the X chromosomes. Nuclei i and ii both show synapsis between a presumed pair of Xs and exclusion of the third X to a distinct domain of DAPI staining. In i, the third X lacks SYP-1 (red arrow). In ii, the third X shows SYP-1 over part of its axis length (yellow arrow), and absence of SYP-1 over the remainder (red arrow); for clarity, top two-thirds of this nucleus is rendered. Frequency of classes is indicated in the schematics. The remaining three nuclei showed all three X chromosome axes together for part of their length. Nuclei from 3 germ lines were analyzed. <b>B.</b> The major class of X chromosome synapsis configuration in triplo-X <i>spo-11</i> nuclei, illustrated as in A. The X chromosomes occupy a single, unitary DAPI domain, indicating that the third X is not excluded; the majority of HTP-3 staining within the X chromosome domain appears to localize to a single SC (yellow triangle). The remaining three nuclei showed partial exclusion of the third X chromosome, which showed some SYP-1 loading. Nuclei from 4 germ lines were analyzed.</p

Relationship between H3K9me2 acquisiton at synapsis defects and loss of SUN-1 phosphorylation in altered karyotypes.

<p><b>A.</b> Relationship between appearance of the H3K9me2 chromatin mark (red) and downregulation of SUN-1 S8-Pi (green) illustrated by IF in whole mount hermaphrodite germ lines of the indicated karyotypes. Green lines are drawn as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen-1003963-g005" target="_blank">Figure 5</a>, and red lines delineate the region in which the majority of nuclei in each row show the specific mid-prophase nuclear staining pattern characteristic of each karyotype (see text). Bar = 10 µm. Insets (right) show H3K9me2 (red) staining patterns in DAPI-stained nuclei (gray) from indicated regions of the corresponding germ lines. Bars = 4 µm. <b>B.</b> Major classes of H3K9me2 (red) and SYP-1 (white) staining observed in late pachytene region nuclei of each karyotype illustrated by 3-D surface renderings; DAPI is shown in blue. In altered karyotypes, intense surface domains of H3K9me2 correspond most commonly to unsynapsed chromatin (red arrows), but sometimes mark synapsed chromatin as well (yellow arrows). <b>C.</b> Half-nuclear projection of late pachytene region from a triploid hermaphrodite germ line stained for H3K9me2 (blue), HTP-3 (red) and SYP-1 (green), revealing differential H3K9me2 association with two classes of synapsis defects—regions where SYP-1 staining was undetectable (arrows, usually marked) and where SYP-1 staining was very weak compared to surrounding SCs (open triangles, rarely marked). Preferential association of H3K9me2 with regions where SYP-1 was undetectable was confirmed by analysis of 3-D image stacks. Bar = 4 µm. <b>D.</b> Quantitation of the percent of the meiotic prophase zone occupied by SUN-1 S8-Pi-positive nuclei, scored and represented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen-1003963-g005" target="_blank">Figure 5B and C</a>, in triplo-X germ lines of a <i>met-2</i> mutant background (8 germ lines scored). Wild type control is the same as presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen-1003963-g005" target="_blank">Figure 5C</a>.</p

Detection of synapsis imperfections marked by H3K9me2 and elevated apoptosis upon depletion of MET-2 during normal diploid female meiosis.

<p><b>A.</b> 3-D rendering of the bottom two-thirds of a late pachytene diploid nucleus stained for H3K9me2 (blue), HTP-3 (red), SYP-1 (green), and DAPI (gray). White box delineates a small internal H3K9me2 domain that marks terminally unsynapsed chromosome axes, highlighted by red dotted traces in merged images. <b>B.</b> Quantitaton of germ cell apoptosis in diploid hermaphrodites (24 h post-L4 at 20°C) of the indicated genotypes. Two-tailed Mann-Whitney tests indicate highly significant differences for <i>wild type</i> vs. <i>met-2</i> (P<0.0001), <i>pch-2</i> vs. <i>met-2; pch-2</i> (P<0.0001) and <i>met-2</i> vs. <i>met-2; spo-11</i> (P = 0.0049) comparisons, but not for <i>spo-11</i> vs. <i>met-2; spo-11</i> (P = 0.847) or <i>met-2</i> vs. <i>met-2; pch-2</i> (P = 0.961). Numbers of gonads scored: wild type, n = 71; <i>met-2</i>, n = 135; <i>pch-2</i>, n = 65; <i>met-2; pch-2</i>, n = 125; <i>spo-11</i>, n = 64; <i>met-2; spo-11</i>, n = 46.</p

Male germ cells containing supernumerary chromosomes show a greater capacity to apply H3K9me2.

<p><b>A.</b> DAPI staining (white) and IF for SUN-1 S8-Pi (green) and H3K9me2 (red) in whole mount male diploid (1X:2A) and triploid (2X:3A) germ lines. Green and red lines are drawn as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen-1003963-g006" target="_blank">Figure 6</a>; gray lines indicate meiotic zone used for scoring in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen.1003963.s008" target="_blank">Figure S8</a>, from onset of meiotic SUN-1 S8-Pi staining through the end of the pachytene stage. Bar = 10 µm. Insets (right) show H3K9me2 (red) staining patterns in DAPI-stained mid- to late pachytene nuclei (gray). Bars = 4 µm. <b>B.</b> Quantitation of the number of H3K9me2 domains in late pachytene nuclei of hermaphrodite vs. male germ lines with a single odd X chromosome (compare 3X:2A vs. 1X:2A), or a triploid karyotype (compare 3X:3A vs. 2X:3A). 3-D-rendered images were used for scoring. Each stacked bar represents the late pachytene region of a single germ line of the indicated genotype, comprising 18–47 nuclei in hermaphrodites and 14–26 nuclei in males; the “>3” category includes nuclei in which contiguous H3K9me2 domains occupied half or more of the nuclear volume. <b>C.</b> Coincidence of H3K9me2 (blue) staining with synapsis defects in triploid hermaphrodites (3X:3A) and males (3X:2A) illustrated by 3-D surface renderings of half-nuclei from the late pachytene region. HTP-3 (red), SYP-1 (green), and DAPI (gray) are shown. Unsynapsed regions (arrows) are marked in both sexes, whereas aberrantly synapsed regions are not marked in the hermaphrodite (open triangle), but robustly marked in the male (filled triangle).</p

Homolog groups associate at the pairing center in trisomic and polyploid <i>C. elegans</i>.

<p><b>A.</b> Schematic depictions of the karyotypes of hermaphrodite worms analyzed in this work. X chromosomes are represented in black, autosome sets in gray; open boxes at one end represent PCs. <b>B.</b> IF for X-PC binding protein HIM-8 (red) and chromosome I- and IV-PC binding protein ZIM-3 (green, see schematic to right) in karyotypes indicated above; DNA is counterstained with DAPI (blue); full nuclear projections of nuclei in the early pachytene region are shown. One prominent HIM-8 focus and two prominent ZIM-3 foci are typically visible in each nucleus. Minor speckles likely reflect localization of PC proteins to additional chromosomal sites outside of the PC <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen.1003963-Nabeshima1" target="_blank">[27]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen.1003963-Phillips3" target="_blank">[61]</a> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen.1003963.s001" target="_blank">Figure S1</a>). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen.1003963.s002" target="_blank">Figure S2</a> for images of full germ lines and quantitation of pairing frequencies at specific time points. Bars = 4 µm.</p

X chromosome territories usually occupy a unitary domain early in the synapsis window.

<p><b>A.</b> Use of S-phase labeling to identify a highly synchronous cohort of meiotic prophase nuclei. Image of the TZ region of a diploid germ line (DAPI in gray) labeled with EdU (red) for 5 h prior to fixation; circles indicate synchronous nuclei displaying a single labeled chromosome pair, <i>i.e.</i>, the late-replicating X chromosomes. Bar = 4 µm. <b>B.</b> 3-D renderings of nuclei early in the synapsis window, illustrating the major class in which PCs are paired and X chromosome territories are “extended” (length greater than twice the width). In all karyotypes the territory comprises a unitary spatial domain suggesting rough alignment. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen.1003963.s003" target="_blank">Figure S3</a> for all classes. Germ lines were injected with fluorescently labeled dNTPs 10 h prior to fixation and HIM-8 staining (green); nuclei displaying labeled X chromosomes are found in the late TZ in these samples. <b>C.</b> Quantitation of the spatial organization of X chromosome territories in nuclei fixed at 10 h post labeling. Categorization is based on X-PC pairing status and labeled X chromosome territory shape as indicated in the schematics. Informative classes are highlighted in green and orange. Nuclei from 4–6 germ lines were analyzed.</p

Homolog groups preferentially achieve pairwise synapsis, but supernumerary chromosomes challenge this process.

<p><b>A.</b> Synapsis patterns in the indicated karyotypes illustrated by half-nuclear projections of nuclei from the mid-pachytene region. HTP-3 (red) marks chromosome axes and SYP-1 (green) marks SC central region; overlap (yellow) indicates synapsed segments with full synapsis illustrated in the diploid. The altered karyotypes display a mixture of nuclei containing unsynapsed regions (arrows) and nuclei exhibiting apparently complete synapsis (circles; assessments were made from full projections). Images are not deconvolved; bars = 4 µm. <b>B.</b> Major classes of X chromosome synapsis configurations in the mid-pachytene region illustrated by 3-D surface renderings of individual nuclei stained for SYP-1 (white) and HIM-8 (green) 24 h after S-phase labeling (red) of X chromosomes. Pairwise synapsis, characteristic of the diploid, also predominates in tetraploid and triplo-X nuclei; whereas in triploids, all three X chromosomes typically occupy a joint domain containing a single SC track. White dotted traces indicate SYP-1 tracks associated with X chromosome territories. iv: region where a single SC track is associated with four X chromosomes (purple arrow), and complex SC tracks associated with the remainder of the X territory (gray dotted traces); v: unsynapsed third X (red dotted trace); vi: short SC track associated with a self-synapsed third X (yellow arrow); vii and viii: single SC track within the domain occupied by all three X chromosomes (blue arrow). Frequency of each synapsis configuration is indicated below each image pair. Nuclei from 4–6 germ lines were analyzed. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen.1003963.s004" target="_blank">Figure S4</a> for full quantitation of classes in triplo-X and triploids; the remaining two tetraploid nuclei appeared to display partial pairwise homologous synapsis with partial asynapsis or partial heterologous synapsis.</p

The Future Xa and Future Xi Exhibit Distinct Frequencies of Singlet FISH Signals

<div><p>(A) Allele-specific FISH for the <i>Xic</i> (red) in Δ <i>Xist</i>/+ ES cells. An <i>Xist</i> probe (green) identifies the wild-type allele. White arrowhead indicates the Δ <i>Xist</i> allele. </p> <p>(B) Allele-specific FISH for the <i>Xic</i> (red) in <i>Tsix</i>–pA/+ ES cells. <i>Tsix</i> RNA (green) identifies the wild-type allele. Grey arrowhead indicates the <i>Tsix</i>–pA allele. </p> <p>(C) Table summarizing scoring of allele-specific FISH in Δ <i>Xist</i>/+ and <i>Tsix</i>–pA/+ ES cells. For three X-chromosomal loci, SD cells were scored for identity of the allele displaying the singlet signal. The X chromosome indicated in black always becomes the Xa, and that in gray and marked with an asterisk always becomes the Xi. The allele indicated in green will be the expressed allele after X-inactivation and the allele indicated in red will be the silent allele. <i>p</i>-Values reflect the probability that the observed distributions are random. </p></div

X Chromosomes Appear to Switch between States

<div><p>(A) The two SD signal configurations observed for <i>Ccnb3</i> (red) by allele-specific FISH in X.2/X^wt ES cells, in which one X chromosome is fused to Chromosome 2. The marked allele (asterisk) is scored by its proximity to a <i>CEN-2</i> probe (green). The <i>CEN-2</i> signal on wild-type Chromosome 2 is indicated by parentheses. </p> <p>(B) Allele-specific scoring of <i>Ccnb3</i> in X.2/X^wt ES and four single cell-derived clones. Nonsignificant <i>p</i>-values indicate a random distribution. </p> <p>(C) Scoring of allele-specific FISH for the <i>Xic</i> ( <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040159#sg002" target="_blank">Figure S2</a>) in two independently derived 129-tet/ <i>cas</i> ES cell lines <b>.</b><i>p</i>-Values indicate that the <i>Xic</i> on the 129 chromosome exhibits a singlet signal at a higher frequency than would be expected by random chance. </p></div

X Chromosomes Differ from One Another in ES Cells

<div><p>(A) Map of the X chromosome showing positions (Mb) of loci assayed for SIAR.</p> <p>(B) Concordant <i>Mecp2</i> (red) and <i>Hprt</i> (green) (left) and discordant <i>Xic</i> (red) and <i>Mecp2</i> (green) (right) FISH signals. </p> <p>(C) Frequencies of concordance and discordance for specified locus pairs in ES cells. <i>p</i>-Values, determined using a χ² test, reflect the probability that the observed distributions are random. </p></div