Requirement for Heterochromatin Stability Figure 2. Su(var)3-9 mutant oocytes and nurse cells display increased DNA damage in heterochromatin. A) The images show cH2Av (white in top panel and red in bottom panel) and C(3)G (green) IF in whole-mount germaria from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. C(3)G is part of the synaptonemal complex and used to distinguish oocytes from nurse cells, both of which contain DSBs. Each image is an optical section; bar = 7 mm. B) and C) The graphs show the average numbers and volumes (relative to total nuclear volumes) of cH2Av foci in nurse cells and oocytes from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. Both quantitation methods showed that cH2Av foci in Su(var)3-9 nurse cells were significantly increased over wild type (p,0.001). cH2Av foci in Su(var)3-9 oocytes were significantly increased over wild type (p,0.001). The numbers of cH2Av foci in mei-W68; Su(var)3-9 nurse cells and oocytes were lower than Su(var)3-9 alone and not significantly different from wild type (p,0.001). Error bars indicate standard deviations, p values were calculated by Student’s t test, and n.15 for each cell type. D) Combined cH2Av IF (red) and satellite FISH (green) in wild-type and Su(var)3-9 germaria; C(3)G (grey) staining identifies the oocytes. Percent of oocyte and nurse cells that displayed overlap between cH2Av and satellite signals are shown. Each image is an optical section, and cells are 5 mm wide. doi:10.1371/journal.pgen.1000435.g002 percentage of each nucleus in mutant cells stained for cH2Av. cH2Av foci can fuse with each other, and thus foci counts can under-represent the phenotypic severity in mutant cells. Quanti- tative volumetric analysis can address this issue, but can also be influenced by varying intensity values in whole-mount IF experiments. The number of cH2Av foci in Su(var)3-9 nurse cells and oocytes were significantly increased over wild type (Figure 2B, p,0.001). Quantitative analysis of cH2Av volumes (relative to nuclear volumes) in wild type and Su(var)3-9 yielded similar results (Figure 2C). Increased DNA damage foci in germaria may be due to higher frequencies of meiotic breaks or defects in repairing meiotic breaks. However, we did not observe any cH2Av signals in the Su(var)3-9 late stage oocytes (data not shown), where meiotic crossover would have completed, suggesting that increased DNA breaks were not due to defective meiotic break repair in mutants. Does the increased DNA damage in Su(var)3-9 mutant oocytes and nurse cells occur in heterochromatin, as observed for somatic cells? The heterochromatic regions in oocytes and nurse cells do not coalesce into clearly-definable regions, and HP1 is mislocalized in Su(var)3-9 cells due to severely reduced H3K9 methylation. Therefore, we performed combined cH2Av IF and FISH with satellite DNA probes in whole-mount wild type and Su(var)3-9 mutant germaria. The probes included the 1.688, AACAC, AATAT, dodeca, AATAG, 1.686, and AAGAG satellites, which correspond to approximately 34 megabases of the heterochroma- tin (Materials and Methods), less than half of heterochromatic PLoS Genetics | www.plosgenetics.org 4 March 2009 | Volume 5 | Issue 3 | e1000435