1. A Component of C. elegans Meiotic Chromosome Axes at the Interface of Homolog Alignment, Synapsis, Nuclear Reorganization, and Recombination 
Florence Couteau, Kentaro Nabeshima, Anne Villeneuve, Monique Zetka 
Current Biology 
Volume 14, Issue 7, Pages (April 2004)
DOI: /j.cub

2. Figure 1
him-3 Mutants Show Defects in the Spatial Reorganization of the Early Prophase Nucleus
(A) Low-magnification images of DAPI-stained germ lines. Prophase progression is from left to right. After premeiotic S phase, nuclei enter the germ line transition zone (underlined), corresponding to the leptotene-zygotene stages of meiosis [4, 18], and undergo a spatial polarization of their contents; the chromosomes asymmetrically cluster to one side of the nucleus, giving rise to a crescent-shaped appearance, while the nucleolus is positioned off-center and opposite to them. This zone is followed by the pachytene region, in which nuclei increase in volume and the synapsed chromosomes disperse about the nuclear periphery and around the centrally positioned nucleolus. Chromosome clustering does not take place in gk149 mutant nuclei, and chromosomes remain dispersed about the nuclear periphery. Nuclear polarization in the me80 germ line is variable; few early prophase nuclei (right) exhibit the tight clustering of chromosomes typical for the transition zone of wild-type germ lines, yet nuclei with a partially polarized appearance are found in the region that would normally correspond to the pachytene stage. In the vv6 mutant, nuclei with a transition-zone-like appearance occupy an expanded portion of the germ line (underlined), and the number of pachytene-like nuclei is decreased. The scale bar represents 10 μm.
(B) High-magnification immunofluorescence micrographs of wild-type and him-3 mutant nuclei from the transition zone region of the germ line (zone II). The nuclear periphery is outlined with α-LAMIN staining (green), and localization of the nucleolus is shown with α-Nop1p (red). Images are projections of a 1.4-μm-thick stack from the middle of the nuclei. Wild-type nuclei show spatial polarization in which the DAPI-stained chromatin (blue) is tightly clustered to one side of the nucleus and the nucleolus is displaced to the opposite side. Nuclei in the gk149 mutant are not polarized; chromatin is widely dispersed about the nuclear periphery and surrounds centrally positioned nucleoli. Nuclei in the vv6 mutant exhibit partial chromosome clustering, but nucleoli remain in a more central position. The scale bar represents 7 μm.
Current Biology  , DOI: ( /j.cub )

3. Figure 2 HIM-3 Is Required for Establishment of Homolog Pairing
Gonads were divided into five equivalently sized intervals along their distal-proximal axes (45–50 μm each) corresponding to the following stages: mitotic/pre-meiotic zone (I), leptotene-zygotene (II), and early, middle, and late pachytene (III–V). We used single-copy probes to monitor pairing for loci at chromosome I left (LG IL), chromosome V left (LG VL), and chromosome X left (LG XL). We also assessed pairing by using a probe targeting the 5S rDNA locus, which represents approximately 15% of chromosome length from the right end of chromosome V.
(A) Table indicating the percent of paired FISH signals detected in each zone in wild-type and homozygous mutant germ lines. The number of scored nuclei for each zone is indicated in parentheses. For the him-3(vv6);syp-1(me17) double mutant, levels of pairing detected in every zone did not differ significantly from those observed in the vv6 single mutant.
(B) Histograms showing pairing levels for the indicated probes in each strain. In the gk149 mutant, pairing levels did not rise significantly above background levels observed in the premeiotic region for any of the probes tested. Pairing in the vv6 mutant was also greatly reduced at all autosomal loci tested, but significant levels of pairing were detected at the 5S rDNA locus in zones IV and V (p = 0.01 and p = 0.02) and at the IL locus in zone V (p = 0.04). The pairing level of XL attained in vv6 mutant was not different from wild-type levels in all zones. In the me80 mutant, pairing did not rise significantly above premeiotic levels for IL or VL loci, but pairing did show significant elevation in zone V for the 5S locus (p = by Fisher's Exact test). The pairing level of XL attained in zones II–IV was not different from wild-type levels, indicating that pairing was efficiently initiated at this locus; however, pairing was significantly reduced in zone V (p = 0.01), suggesting that stabilization was not as effective in me80 as in the wild-type.
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4. Figure 3 Synapsis Is Defective in him-3 Mutants
In wild-type germ lines, HIM-3 is first detected in transition zone nuclei at the assembling chromosome cores. HIM-3 loading is then followed by recruitment of SYP-1, and by the pachytene stage of meiosis, overlapping anti-HIM-3 and anti-SYP-1 signals are detected as long contiguous stretches lying at the interface between pairs of parallel DAPI-stained tracks that correspond to pairs of synapsed chromosomes.
(A) SYP-1 loading correlates with the level of HIM-3 recruitment to chromosome cores. Immunofluorescence micrographs represent projections of three-dimensional data stacks that encompass entire nuclei from mid-pachytene (zone IV). In vv6 mutants, wild-type like levels of HIM-3 associate with chromosome cores, but SYP-1 staining is reduced. In me80 mutants, both the loading of HIM-3 and SYP-1 are highly reduced, and in gk149 mutants HIM-3 is not detectable and SYP-1 staining identifies nuclear aggregates of the protein. The scale bar represents 4 μm.
(B) Chromosomes undergo nonhomologous synapsis in vv6 mutants. FISH analysis of pairing levels in vv6 mutant nuclei from zone IV and subsequent α-SYP-1 staining: DAPI (blue), 5S rDNA (red), and SYP-1 (green). In the top nucleus the paired chromosomes V are associated with a SYP-1 tract. In the other nucleus, two FISH signals indicate that chromosomes V are not paired; however, one of these signals lies adjacent to a SYP-1 staining chromosome. The scale bar represents 2 μm.
(C) Graphic representation of the proportion of zone IV nuclei with unpaired 5S rDNA foci in association with SYP-1 (104/147 observed nuclei), with paired foci in association with SYP-1 (25/147), and with unpaired foci with no associated SYP-1 tracts (18/147). The first class comprises three cases: unpaired FISH signals where one signal is on a SYP-1 tract (51%), unpaired signals when both signals are on separate tracts (37%), and unpaired signals separated on the same tract (12%).
Current Biology  , DOI: ( /j.cub )

5. Figure 4
RAD-51 Foci Persist in him-3 Hypomorphs but Not in the Null Mutant
Gonads were divided into five equally sized zones as for time-course analysis of pairing. Scatter plots show the distribution of RAD-51 foci in wild-type and mutant nuclei having at least one RAD-51 focus (every single positive nucleus is represented). The percentage of nuclei having zero RAD-51 foci and the total number of nuclei scored (in parentheses) are indicated at the right top corner of each panel. Neither the number of RAD-51-positive nuclei in zone II of gk149, vv6, and me80 nor the distribution of RAD-51 foci within these nuclei is different from that of the wild-type in zone II (gk149: p = 0.09, vv6: p = 0.37, me80: p = 0.59, two-tailed Mann-Whitney test, 95% C.I.), suggesting that recombination was initiated at the same frequency in the wild-type and him-3 mutants. In gk149 mutants, the distribution of RAD-51 foci is not different from that of the wild-type in zones III and V (p = 0.61, and p = 0.16 respectively); in both cases, RAD-51 foci peak in zone III (average of 4.1 foci per positive nucleus in gk149 mutants and 3.4 foci in the wild-type) and are drastically reduced in zone V (average of 1.7 foci per positive nucleus in gk149 mutants and 1.6 in the wild-type). In zone IV the distribution shows a significant difference (p = 0.003), however; the average number of RAD-51 foci in the positive nuclei is 2.6 in gk149 mutants and 2.3 in the wild-type. In contrast, RAD-51 foci distribution in both vv6 and me80 mutants is extremely different from that in the wild-type in zones III–V (p < in both cases); in both mutant backgrounds, RAD-51 foci levels peak in zone IV, where the average number of foci in positive nuclei is 7.4 and 10.3, respectively. In zone V, 67% and 57% of me80 and vv6 nuclei still show RAD-51 foci, with an average of 3.4 and 4.8 and foci per nucleus, respectively, whereas less than 16% of WT and gk149 nuclei show RAD-51 foci at this stage.
Current Biology  , DOI: ( /j.cub )