DTC architecture and the plexus region.

<p>(A) The distal gonad includes the DTC niche (red) and the mitotic zone. Yellow circles represent germ cells in the mitotic cell cycle; green circles are germ cells in meiotic S-phase; green crescents are germ cells in early meiotic prophase when chromosomes have begun to pair. Germ cells are connected by a cytoplasmic core. The mitotic zone consists of a distal stem cell pool (GSC pool) adjacent to the niche. As germ cells leave the GSC pool and progress proximally through the mitotic zone they mature. At the proximal edge of the mitotic zone, germ cells progress into early meiotic prophase as they move into the transition zone (TZ). (B) Germ cell position is scored in Hoechst-stained extruded gonads as number of germ cell diameters (gcd) from the distal end. The DTC nucleus (DTC) is oval (red oval); germ cell nuclei are round (white circles). (C) P<i>_lag-2::myr-GFP</i> (myr-GFP, green) allows visualization of DTC membranes. This view through the central region of the gonad shows the DTC capping the distal end (cap) and short intercalating processes (SIPs; red arrowheads) on either side of germ cell nuclei (Hoechst, blue). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088372#pone-0088372-g001" target="_blank">Figure 1C</a> modified from Byrd and Kimble <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088372#pone.0088372-Byrd1" target="_blank">[9]</a>. (D) This “core” projection of central sections (10 µm) of a confocal z-series shows the DTC plexus, which includes the cap, short intercalating processes (SIPs; red arrowheads), long external processes (distal LEPs; green arrowheads) and internal fragments (white arrowheads). (E) The DTC cap and LEPs are best visualized in a full projection of a z-series. The cap and LEPs are detected quantitatively using Plot profile. The graph below shows pixel intensity (y-axis) along the distal-proximal axis (x-axis). Dotted lines mark the distal end of the gonad and the proximal boundary of the cap (end of initial peak of GFP fluorescence) or LEPs (first position where pixel intensity reaches background). (F) The DTC plexus is best visualized in a “core” projection of the central ten 1 µm slices of a z-series. The plexus is detected quantitatively using Plot profile. The graph below shows pixel intensity (y-axis) along the distal-proximal axis (x-axis). Dotted lines mark the distal end of the gonad and the proximal boundary (first position where the pixel intensity reaches background) of the plexus. (B–F) Scale bars = 10 µm. (G) DTC features measured by three different methods give similar results. First, live animals were scored by eye using wide-field fluorescent microscopy. Second, dissected gonads were scored by eye using wide-field fluorescent microscopy. Third, dissected gonads were scored quantitatively using confocal microscopy and Plot profile. Bar graphs show average lengths of DTC features. Error bars indicate standard deviation.