A DTC niche plexus surrounds the germline stem cell pool in Caenorhabditis elegans

Abstract

The mesenchymal distal tip cell (DTC) provides the niche for Caenorhabditis elegans germline stem cells (GSCs). The DTC has a complex cellular architecture: its cell body caps the distal gonadal end and contacts germ cells extensively, but it also includes multiple cellular processes that extend along the germline tube and intercalate between germ cells. Here we use the lag-2 DTC promoter to drive expression of myristoylated GFP, which highlights DTC membranes and permits a more detailed view of DTC architecture. We find that short processes intercalating between germ cells contact more germ cells than seen previously. We define this region of extensive niche contact with germ cells as the DTC plexus. The extent of the DTC plexus corresponds well with the previously determined extent of the GSC pool. Moreover, expression of a differentiation marker increases as germ cells move out of the plexus. Maintenance of this DTC plexus depends on the presence of undifferentiated germ cells, suggesting that germ cell state can influence niche architecture. The roles of this DTC architecture remain an open question. One idea is that the DTC plexus delivers Notch signaling to the cluster of germ cells comprising the GSC pool; another idea is that the plexus anchors GSCs at the distal end.

Figure 1. DTC architecture and the plexus region.

(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_lag-2::myr-GFP (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). Figure 1C modified from Byrd and Kimble . (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.

Figure 2. The DTC plexus ends where the differentiation marker, GLD-1, increases.

(A) Core confocal projection of an extruded gonad showing the DTC plexus region (red) and GLD-1::GFP (green). The position where GLD-1 becomes easily detectable is marked by the dotted line. (B) Pixel intensity plots of myr-tdTom (red line) and GLD-1::GFP (green line). GLD-1::GFP intensity increases where myr-tdTom intensity drops to background, midway through the MZ. (C) Hoechst-stained germ cell chromatin (blue) shows proximal edge of MZ (dashed line). (D) Six additional germlines were scored using Plot profile as in (B). All 6 are similar to (B): myr-tdTom intensity drops to background where GLD-1::GFP intensity increases. (E) Average positions of DTC plexus and GLD-1::GFP (gcd±standard deviation). Scale bars in (A) and (C) = 10 µm.

Figure 3. DTC architecture depends on germ cell state.

(A–E) Extruded gonads from animals expressing myr-tdTom (A-C) or c-GFP (D,E). All fluorescence was pseudo-colored green for simplicity. Genotypes and growth conditions are shown in first column (see Methods for details); GLP-1/Notch activity is summarized in second column; germ cell state in the niche region is summarized in the third column (yellow, undifferentiated germ cells; green, germ cells in early stages of meiosis). Scale bar = 10 µm.

Figure 4. The DTC plexus forms in adults and is maintained through the reproductive period.

(A–C) Extruded gonads from animals expressing myr-GFP. (A) L4. Pre-reproductive larva. These animals make only sperm; they do not produce embryos. The DTC has both leader and niche function at this stage and germ cell number is increasing. DTC architecture includes the cap and a few SIPs found associated with the cap. LEPs are not observed. (B) Reproductive adult, 24 hours after L4. The DTC functions as a stem cell niche . Germ cell number is maintained. DTC architecture includes cap, extensive plexus and LEPs. (C) Post-reproductive adult, 96 hours after L4. Germ cell number is maintained and DTC architecture is similar to that seen in reproductive adults. Scale bar = 10 µm.