DSB-2 localizes to chromatin in early meiotic prophase nuclei, concurrent with DSB marker and SUN-1 Ser8-phosphorylation.

<p>(A) Immunofluorescence image of a WT hermaphrodite gonad (from distal tip to end of pachytene) stained with DAPI and antibodies that recognize DSB-2 and SUN-1 S8P. In this and all subsequent figures, the distal tip (which contains mitotically cycling germ cells) is at the left, and meiotic progression is from left to right. DSB-2 first appears on chromatin at meiotic onset in transition zone nuclei (corresponding to the leptotene/zygotene stages of meiotic prophase) and disappears around mid-pachytene stage of meiotic prophase, with a few outlier nuclei retaining DSB-2 in later pachytene. Here and in subsequent figures, yellow lines demarcate the “DSB-2-positive” zone and a cyan line marks the end of the pachytene zone. Co-staining shows correlation between DSB-2-positive and SUN-1 S8P-positive meiotic nuclei. (Note: SUN-1 S8P is also present in a few pre-meiotic nuclei; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003674#pgen.1003674-Penkner1" target="_blank">[23]</a>). A close-up of the large box is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003674#pgen-1003674-g005" target="_blank">Figure 5C</a>. Scale bar, 15 µm. (B) Close-up of nuclei outlined by the small box in (A). DSB-2 localizes to a few bright patches/foci, as well as fainter stretches/foci along the entire chromatin (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003674#pgen-1003674-g005" target="_blank">Fig. 5C</a>). As nuclei reach mid-pachytene, the DSB-2 signal becomes fainter (narrow arrowhead), however in some nuclei signal gets brighter along most of chromatin (broad arrowhead). (C) Immunofluorescence image of a WT hermaphrodite gonad from entry into meiotic prophase to mid-to-late pachytene, stained with antibodies that recognize DSB-2 and RAD-51. RAD-51 foci (marking processed DSBs) appear in nuclei shortly after DSB-2 staining appears on chromatin upon meiotic entry, and the RAD-51 foci disappear shortly after DSB-2 is no longer present on chromatin in mid-pachytene nuclei. Inset shows that RAD-51 foci mostly do not co-localize with concentrated DSB-2. DSB-2-bright outlier nuclei in late pachytene contain high levels of RAD-51 foci. Scale bar, 15 µm. (D) Immunofluorescence image of the early mid-pachytene to late pachytene region of a WT hermaphrodite gonad expressing GFP::COSA-1 (strain AV630), stained with antibodies that recognize DSB-2 and GFP. COSA-1 foci marking designated CO sites appear in nuclei only after the removal of DSB-2 from chromatin; DSB-2-bright outlier nuclei in the late pachytene region lack COSA-1 foci, even when COSA-1 foci are present in neighboring nuclei. Close-ups are shown in insets. Scale bar, 15 µm.