Fig. 1. -HIS2AV staining in wild-type female meiosis. (A) Projection of an image stack containing a single germarium stained for ORB (green), -HIS2AV (red) and DNA (blue). Before region 2a, four incomplete premeiotic divisions produce a cyst of 16 cells interconnected by ring canals. Because developing cysts are propelled to the posterior end of the germarium (in direction of arrow), they are usually arrayed in order of their relative developmental ages (Carpenter, 1975a). The ORB protein (Lantz et al., 1994) was used as a cytoplasmic marker to identify the 16-cell cysts, which are numbered in order of developmental stage. When cysts with two pro-oocytes are initially formed, they are in region 2a (cysts 1-4) and have a round shape. This region contains early zygotene and pachytene oocytes. Several cells within each cyst show evidence of entering meiosis, and two cells, the pro-oocytes, have four ring-canals and reach pachytene with complete assembly of synaptonemal complex (SC) (Carpenter, 1975a). One of these cells will become the oocyte. When the cysts flatten out and become encircled by follicle cells, they are in region 2b (cyst 5 and 6) and 3 (cyst 7). Region 3 oocytes are in mid-late pachytene. ORB becomes enriched in the oocyte in regions 2b and 3 (arrows, cysts 5-7), but initially (more anterior) it appears uniformly in all 16 cells of a cyst (region 2a, cysts 1-4). In projections such as this, it is difficult to determine which nuclei contain foci. It is clear from this view, however, that abundant -HIS2AV foci are not observed until cysts 3. C(3)G staining is usually present in the first ORB staining cysts (1 and 2) (Page and Hawley, 2001) (data not shown), suggesting that -HIS2AV staining appears after SC formation (see Fig. 2). Single optical sections show -HIS2AV foci in nuclei from cyst 3 (B) and cyst 5 (C) but not later stage nuclei from cysts 6 and 7 (D). Arrowheads mark the oocytes. Bar, 10 µm.