Molecular analysis of dpy-28. (A) Corresponding genetic and physical maps of chromosome III near dpy-28. dpy-28 was mapped to the interval between markers cul-1 (on W04F9) and vab-7 (on M142) and then between RFLPs (asterisks) on cosmids K01G5 and K11D9. (B) RNAi assay identifying dpy-28. Candidate dpy-28 RNAs were injected into sdc-3(Tra) unc-76/++ XX animals, and F1 Unc progeny were scored for hermaphrodites. F1 Unc homozygous sdc-3(Tra) unc-76 XX progeny from untreated heterozygous mothers develop as pseudomales. This transformed (Tra) phenotype is reversed by dpy-28 mutations (DeLong et al. 1993). RNA corresponding only to cDNA yk32e9 suppressed the Tra phenotype and caused all phenotypes typical of dpy-28 mutants. (C) dpy-28 gene structure. Promoter, exons, and introns are represented by a horizontal line, boxes, and diagonal lines, respectively. Molecular changes in four dpy-28 alleles are shown. DPY-28 antibodies were made against amino acids 351–771 (black bar). (D) Western blot of extracts from wild-type and dpy-28(s939) embryos probed with DPY-28 antibodies. DPY-28 migrates at ∼160 kDa and is undetectable in s939 embryonic extract. (E) Western blot of lysates from wild-type and dpy-28 gravid adult hermaphrodites probed with antibodies to DPY-28 and β-tubulin, the loading control. Changes in DPY-28 level and size are consistent with molecular lesions in the mutants. (F) DPY-28 coimmunoprecipitates with known members of the DCC. Reciprocal coimmunoprecipitation of DPY-26, DPY-27, DPY-28, and MIX-1 with antibodies to each of the four proteins indicates that DPY-28 is a subunit of the DCC.

DPY-28 associates with X chromosomes of XX but not XO embryos. (A) Immunolocalization of DPY-28 in XX embryos. Shown are false-color confocal images of wild-type and dpy-28 mutant XX embryos costained with DPY-28 antibodies (green) and the DNA-intercalating dye DAPI (red). Overlap of DPY-28 and DAPI appears yellow in merged images. An enlarged, merged nucleus is shown for each genotype. The diffuse nuclear DPY-28 pattern in young embryos and the punctate DPY-28 pattern in older embryos with ≥40 cells are absent in dpy-28(s939) or dpy-28(y283) mutants. (B,C) DPY-28 associates with X chromosomes of XX but not XO embryos. (B) DPY-28 colocalizes with X-bound DPY-26 in wild-type XX embryos. (C) A him-8; yIs34(Pxol-1∷gfp) XO embryo with >40 cells costained with antibodies against DPY-28 (green) and XOL-1 (red). XOL-1∷GFP is expressed exclusively in XO embryos, where DPY-28 appears diffuse nuclear. (D) DPY-28 (red) associates with mitotic chromosomes of young embryos prior to the onset of dosage compensation. DPY-28 colocalizes with DAPI on mitotic chromosomes, in contrast to mitotic condensin subunit MIX-1 (green), which colocalizes with centromeres (Hagstrom et al. 2002). (E) Association of DPY-28 with X requires other dosage compensation proteins. Shown are embryos with null mutations in various dosage compensation genes costained with DPY-28 antibodies (green) and DAPI (red). Bars, 5 μm.

DPY-28 accumulates in germline nuclei, and dpy-28 mutants are defective in mitotic chromosome segregation but progress normally through meiotic prophase. (A) Diagram of adult hermaphrodite gonad arm. Each arm is a U-shaped tube that produces male and female gametes. Germ nuclei proliferate in the distal premeiotic zone, then enter meiosis and progress through meiotic prophase stages as they move proximally. In the transition zone, homologs pair and initiate synapsis. Synapsis is complete prior to pachytene, and recombination ends during pachytene. Disassembly of the SC and condensation of homologs by condensin II begin at pachytene exit. At diakinesis, each oocyte nucleus has six DAPI-stained bodies that correspond to six pairs of recombined homologs. (B,C) DPY-28 accumulates in wild-type but not dpy-28(y402) mutant germlines. Shown are regions of hermaphrodite gonads stained with DAPI (red) and DPY-28 antibodies (green). In wild-type gonads, DPY-28 accumulates in premeiotic nuclei and remains nuclear in the meiotic transition zone, partially coincident with DAPI. In pachytene, DPY-28 concentrates around the nuclear periphery, where meiotic chromosomes reside. (D) FISH analysis of gonads stained with DAPI (red) and FISH probes 5S rDNA or X locus (green). Premeiotic nuclei of wild-type and dpy-28(s939) gonads typically have two FISH signals, indicating normal ploidy, but dpy-28 mutants have occasional macronuclei (arrow) with multiple FISH signals, indicating aneuploidy or polyploidy. Wild-type and dpy-28 mutant nuclei have two FISH signals in early transition zone before homolog synapsis and one signal in mid- to late transition zone and in pachytene, indicating normal homolog synapsis. Premeiotic and transition zones (shown in full length) are of equivalent length in wild-type and dpy-28 mutants. The dashed white lines separate a single row of premeiotic nuclei from transition zone nuclei. Bars, 5 μm.

dpy-28 mutations alter the number and distribution of COs on X chromosomes and autosomes. (A–C) CO analysis of chromosomes X, I, and II in wild-type and dpy-28 mutant animals using snip-SNPs. Physical and genetic map positions of the snip-SNP markers (red) are shown above the CO frequency chart. CO frequencies (yellow numbers in boxes) were calculated by assessing recombination in the intervals between snip-SNPs and applying the formula (number of COs in the interval)/(total meiotic products assayed). Shown are the number of triple-crossover (3-CO), double-crossover (2-CO), single-crossover (1-CO), and noncrossover (0-CO) chromatids and total assayed chromatids (n). (%) Percentage of 0-COs by the formula 100(0-CO/n). The relative recombination frequencies (mutant/wild type) are indicated by color tags. Yellow reflects the greatest increase and black reflects the greatest decrease. (A) Heterozygous and homozygous dpy-28 mutations s939, y283, or y402 affect meiotic crossing over on X. Five intervals between snip-SNP markers A–F were assayed for recombination. For all dpy-28 mutant alleles, CO frequencies were elevated in the right half and depressed in the left half. Also, 2-COs and 3-COs occurred in heterozygous and homozygous s939 and y402 animals. Map expansion and contraction is diagrammed in Supplemental Figure 2. (B) s939/+, y283/+, and y402/+ exert a milder effect on crossing over on chromosome I versus X. Four intervals between snip-SNP markers A–E were assayed. (*) For analysis of y402/+ animals, marker B* (genetic position, −12.3) was used instead of B. The wild-type CO frequency of the A–B* interval is 0.04 and the B*–C interval is 0.03. The relative CO frequency in y402/+ animals is compared with these values. (C) s939/+, y283/+, y402/+, and y284/+ cause 2-COs on chromosome II. Five intervals between snip-SNP markers A–F were assayed. See the Supplemental Material for details.

The axes and SC appear wild type in dpy-28 mutants. (A) In false-color confocal images of dpy-28(y283) pachytene nuclei, the axis component HIM-3 (green) and the SC component SYP-1 (red) colocalize (yellow merge) along the entirety of homologs, as in wild-type controls. Bar, 5 μm. (B) Sections of SC in pachytene nuclei of wild-type and dpy-28(s939) gonads visualized by transmission electron microscopy. In both genotypes, the SC appears continuous, and the transverse filaments between lateral elements appear as rung-like structures. Bars, 200 nm. (C) A schematic of the SC structural components and chromatin.

dpy-28 mutants show an increase in DSB-dependent early recombination intermediates but normal progression of these RAD-51 foci. (A) Histograms depicting quantitation of RAD-51 foci in wild-type and dpy-28(s939) germlines. Each column color represents a class of nuclei with the indicated number of RAD-51 foci. The Y-axis shows the percentage of nuclei in each class. The X-axis shows the position along the germline. (M) Premeiotic zone; (TZ) transition zone; (P1) early pachytene; (P2) mid-pachytene; (P3) late pachytene; (n) number of nuclei scored. P-values from χ² tests show that the distribution of RAD-51 foci is different in wild-type and mutant germlines, with a significant increase in P1 and P2 of s939. (See the Supplemental Material for details). (B,C) The increase in RAD-51 foci in dpy-28 mutants requires the DSB enzyme SPO-11. Shown are high-resolution images of wild-type and mutant early to mid-pachytene nuclei stained with antibodies to RAD-51 (green) and the axis protein HTP-3 (red). (C) Enlarged nuclei from B. Bars, 1 μm.

dpy-28 mutants require MSH-5 and SYP-2 for the resolution of RAD-51-bound recombination intermediates and HIM-3 for the increase in RAD-51 foci. All panels are false-color confocal images of germline nuclei stained with DAPI (red) and RAD-51 antibodies (green). (A,B) SC core component SYP-2 and mismatch repair protein MSH-5 are required for the timely removal of RAD-51 foci in wild-type and dpy-28 animals. In syp-2 and msh-5 single mutants and in dpy-28; syp-2 and dpy-28; msh-5 double mutants, RAD-51 foci persist into late pachytene, indicating impaired resolution of meiotic recombination intermediates (Fig. 6). In double mutants, the number of RAD-51 foci is elevated, as in dpy-28 mutants, suggesting that more meiotic DSBs are made in dpy-28 mutants, and their repair requires SYP-2 and MSH-5. (C) Axis component HIM-3 is required in dpy-28 mutants for the increase in RAD-51 foci. dpy-28; him-3 double mutants have a similar number of RAD-51 foci as him-3 mutants. Without HIM-3, dpy-28 mutations do not cause an increase in meiotic DSBs. Quantification for A–C is in Supplemental Figure 4. Bars, 5 μm.

The CO and DSB defects of him-17(null) mutants are suppressed by dpy-28(s939). (A) DAPI-stained oocyte nuclei in late diakinesis from wild-type, him-17(ok424), or dpy-28(s939); him-17(ok424) animals. Each panel shows a set of chromosomes from one oocyte. The wild-type oocyte has six bivalents, indicating normal chiasma formation. Most him-17 oocytes have 12 univalents, reflecting a lack of chiasmata. Most dpy-28; him-17 oocytes have seven DAPI-stained bodies, including five bivalents and two univalents. Bar, 5 μm. (B) Karyotype analysis of diakinesis-stage oocytes. him-17(ok424)^a denotes the him-17(ok424) strain reisolated from the dpy-28; him-17 strain. (C) Quantitation of RAD-51 foci in him-17 and dpy-28; him-17 germlines shows increased foci in double mutants. P-values from χ² tests indicate that the distribution of RAD-51 foci is different in wild-type and mutant germlines. Each column color represents a class of nuclei with the indicated number of RAD-51 foci. The Y-axis shows the percentage of nuclei in each class and the X-axis shows the position along the germline. (M) Premeiotic zone; (TZ) transition zone; (P1) early pachytene; (P2) mid-pachytene; (P3) late pachytene.