Germ Cell Numbers Maintained in Adults
To ask whether the number of germ cells is stable during adulthood, we counted germline nuclei in DAPI-stained animals on 6 consecutive days of adulthood, beginning with young adults 1 d past the fourth larval stage (L4). Although germ cells are continuously lost during this interval to gametogenesis and cell death (Gumienny et al., 1999
), the total number of germ cells was essentially constant: day 1, 919 ± 96 (n = 8); day 2, 869 ± 72 (n = 11); day 3, 1140 ± 127 (n = 5); day 4, 892 ± 91 (n = 6); day 5, 1013 ± 102 (n = 4); and day 6, 1038 ± 113 (n = 4). Therefore, the germ line must be continuously replenished by stem cells. The number of germ cells within the mitotic region was also essentially constant during the same interval: day 1, 243 ± 25 (n = 8); day 2, 232 ± 22 (n = 11); day 3, 227 ± 16 (n = 5); day 4, 222 ± 35 (n = 15); day 5, 214 ± 34 (n = 9); and day 6, 238 ± 12 (n = 4). Cells within the mitotic region must self-renew as they are not depleted during this period of germline maintenance. Together, these results confirm the existence of stem cells in the adult germ line.
S-Phase and M-Phase Indices in the Mitotic Region
In certain vertebrate tissues and in plant meristems, stem cells appear to be slow cycling or quiescent (Zhang et al., 2003
; Passegue et al., 2005
; Stahl and Simon, 2005
). To begin to explore the idea that similar cells might exist within the C. elegans
germline mitotic region, we first used BrdU labeling to detect nuclei in S-phase, a common marker of progression through the cell cycle. Our first BrdU experiment assessed S-phase index (also called labeling index), which refers to the percentage of nuclei in S-phase. Specifically, we exposed young adult hermaphrodites (24 h past the L4 stage) to BrdU for 15 min, prepared their germ lines without any appreciable chase, and then scored the number of S-phase nuclei using BrdU-specific antibodies and total number of nuclei with the TO-PRO-3 DNA dye. Each nucleus was assigned a position according to its distance from the DTC, measured in cell diameters along the distal-proximal axis (B, bottom). For example, all germ cells directly adjacent to the DTC were assigned to row 1 and so forth (B). We counted BrdU-positive and TO-PRO-3–positive nuclei in 2-row intervals and then graphed the percentage of nuclei in S-phase with respect to position (A). In rows 1–16, ~50% of nuclei were labeled, demonstrating that about half of the nuclei were in S-phase at any given time (A and A). Because nuclei in row 1 were of particular interest, being located immediately adjacent to the DTC, we also scored row 1 on its own and found it to have a BrdU-labeling index similar to that of rows 1 and 2 combined (40 ± 16 vs. 43 ± 15%, p = 0.72). The average labeling index in rows 1 and 2 was not significantly different from the average of rows 3–10 (43 ± 15 vs. 55 ± 6%, p = 0.13); however, the labeling index was more variable in rows 1 and 2 (8–71%, n = 12) than in rows 3–10 (38–76%, n = 12). More proximally (rows 11–20), the labeling index decreased until no BrdU-labeled nuclei were seen after row 20 (A).
Figure 2. S- and M-phase indices scored in hermaphrodites 24 h past L4. Solid line indicates region of mitotic cell cycle in all germ lines. Dotted line spans positions of MR/TZ boundaries in all germ lines scored. Dashed line indicates region of meiotic prophase (more ...)
As an alternate measure of mitotic cycling, we examined the percentage of nuclei in M-phase (mitotic index). To this end, we stained M-phase nuclei with anti-PH3 antibodies (Hendzel et al., 1997
) and all nuclei with DAPI or TO-PRO-3 DNA dyes. The mitotic index was ~3.5 ± 1% in rows 1–16, ~0.4 ± 0.3% in rows 17–23 and 0% in more proximal rows (n = 102; B). The mitotic index of rows 3–10 appeared somewhat higher than rows 1–2 or rows 10–16 (4.3% in rows 3–10 vs. 2.9% in rows 1–2, p = 0.04; and 2.7% in rows 11–16; p = 4 × 10−5
). These experiments can be interpreted to indicate that either the cell cycle is ~1.5 times longer or M-phase ~1.5 times shorter in rows 1 and 2 (see below for additional data). A lower mitotic index in the distal-most germ cells has also been reported with a larger data set (Maciejowski et al., 2006
The S- and M-phase indices in Figure 2 were graphed with respect to distance from the DTC. We next graphed the same data sets relative to the boundary between mitotic region and transition zone (MR/TZ). These alternative graphs were done because the position of the MR/TZ boundary varies from germline to germline (A). We assigned negative numbers to rows distal to the boundary and positive numbers to rows proximal to the boundary (, B and C). Labeling index was ~50% in the six rows of germ cells distal to the MR/TZ boundary, dropped to ~25% in the first two rows of the transition zone, and continued to decrease in the next 4 rows (B). By contrast, the mitotic index dropped in the 3 to 4 rows just distal of the transition zone and remained low 3 rows into the transition zone (C). We conclude that the S-phase index is equivalent throughout the mitotic region (about half the nuclei are in S-phase at any position) but that the M-phase index drops dramatically in the proximal-most rows of the mitotic region. The most likely explanation is that many of the S-phase germ cells in the few rows distal to the MR/TZ boundary, as well as those in the transition zone, are in premeiotic S-phase. Thus the transition from the mitotic cell cycle to the meiotic cell cycle occurs over multiple cell diameters (see also Hansen et al., 2004a
Figure 3. Labeling and mitotic indices normalized to MR/TZ boundary from data sets in Figure 2. (A) Percent germ lines with MR/TZ boundary at a given position. (B and C) Labeling index and mitotic index were calculated with respect to the first row of the transition (more ...)
No Quiescent Germline Nuclei in the Mitotic Region
To ask whether all germline nuclei within the mitotic region could be labeled with sufficiently long exposure, we exposed adults to BrdU for increasing time. We began BrdU treatment at 24 h past the L4 stage, as done in the previous experiment, but extended the exposure by 2-h intervals. Because the 4 rows preceding the MR/TZ boundary had a lower mitotic index and are likely to be in premeiotic S-phase (see above), we focused on the distal 16 rows for this experiment, which we refer to as the high mitotic index region. Within that high mitotic index region, the percentage of BrdU-labeled nuclei increased progressively from ~50% after 15 min (A), to ~75% after 4 h (B), to 100% after 8–12 h (C) of BrdU treatment (D). After 8 h, three of eight germ lines possessed 100% labeled germline nuclei, although some nuclei were only partially labeled (C, inset), which we interpret as having spent less time in S-phase. After 12 h, all germ lines had 100% BrdU-positive nuclei (n = 4). Therefore, within an 8–12-h period, all cells within the high mitotic index region had entered or progressed through S-phase.
To ask whether any difference in complete labeling time could be discerned between germ cells at distinct locations within the high mitotic index region, we graphed the increase in S-phase index with increased BrdU exposure times for each of three positions along the proximal-distal axis (E). All had 100% BrdU-positive nuclei within an 8–12 h period (E). Indeed, the time course for rows 1 and 2 was similar to that for rows 3–16. We conclude that no quiescent nuclei are present in the mitotic region and that cell cycles are similar throughout the region.
Estimate of Cell Cycle Length in the Mitotic Region
The data in Figures 2A and 4D permit an estimate of the average cell cycle length for germ cells within the mitotic region. S-phase appears to take roughly half of the cell cycle, because at any one time, about half the germ cells take up BrdU. Furthermore, the interval spanning G1, G2, and M can be estimated from the 8–12 h necessary to obtain >99% BrdU incorporation (Aherne et al., 1977
). Taking these data together, we estimate the length of the mitotic cell cycle to be 16–24 h for germ cells in the high mitotic index region of the adult hermaphrodite germ line. This timing contrasts with cell cycle length during the proliferative phase of germline development, which averages ~4 h (Kipreos et al., 1996
No Label-retaining Cells in the Germline Mitotic Region
In some systems, stem cells are defined by their ability to retain BrdU label after a long chase (Braun and Watt, 2004
; Fuchs et al., 2004
; Potten, 2004
). To learn whether the C. elegans
adult germ line might contain such “label-retaining” cells, we exposed animals at varying stages to extensive BrdU pulses (e.g., 24–48 h), which were followed with increasingly long BrdU-free chases (A). For each experiment, the level of labeling decreased uniformly from all cells within the germline mitotic region (B). , C–G, shows a representative set of germ lines, taken from animals whose labeling regimen began when they were between the L2 and L3 stages and ended 36–48 h later, when they became adults 24 h past the L4 stage. BrdU was therefore incorporated during the larval proliferative phase of germline development, when the cell cycle averages ~4 h in length (Kipreos et al., 1996
). Immediately after the pulse (0-h chase), all germline nuclei in both mitotic and meiotic regions were fully labeled, including oocytes (unpublished data). After a 12-h chase, BrdU staining had decreased within the mitotic region, although all nuclei remained labeled (D). After 24-, 36-, and 48-h chases, BrdU staining decreased more and more, but speckles remained associated with nuclei in the mitotic region (, E and F, unpublished data). As a control, we examined germ lines from animals that had not been treated with BrdU and found small background speckles that did not colocalize with nuclei (H).
The progressive reduction of BrdU staining in the mitotic region appeared essentially uniform along the distal-proximal axis, but BrdU staining remained high in meiotic nuclei. We suggest that BrdU loss from the mitotic germ line results from at least two factors: dilution by replication in the absence of BrdU and movement of germ cells from the mitotic region into meiotic zones (, E–G; see below). The speckles that remained associated with nuclei in the mitotic region even after a 48-h chase are likely to represent chromosomal segments that were retained by chance. We did not notice a reproducible pattern from germ line to germ line; in particular we did not see more BrdU labeling in the distal-most 1 or 2 rows of mitotic germ cells relative to the more proximal mitotic germ cells. In other systems, label-retaining nuclei are easy to detect based on their high levels of BrdU compared with nuclei in surrounding cells (Braun and Watt, 2004
; Potten, 2004
); such nuclei were not observed. We conclude that the C. elegans
germ line does not contain label-retaining nuclei and that BrdU turnover is essentially uniform in all cells within the mitotic region.
Germ Cells in the Mitotic Region Move Proximally into the Meiotic Region
We next focused on movement of BrdU-labeled nuclei from the mitotic region into meiotic zones. To this end, adults (24 h past L4) were fed BrdU for 30 min, and the position of BrdU label was determined during the course of a 24-h chase. Immediately after the 30-min BrdU pulse, BrdU-positive nuclei were scattered throughout the mitotic region in a variable pattern similar to that observed after a 15-min pulse (A and 6A). After a 12-h chase, intensely labeled BrdU-positive nuclei had just moved into the pachytene region, ~12 rows past the MR/TZ boundary (B), and after a 24-h chase they had moved yet more proximally into the pachytene region, ~27 rows past the MR/TZ boundary (C). To estimate the rate of movement, we scored the proximal border of intensely labeled nuclei (inverted triangle in , A–C), which moved proximally during the 24-h chase (, A–D). On average, the border moved at approximately 1 row per hour (D). In these same germ lines, we also saw uniform loss of BrdU label from germ cells within the mitotic region (, A–C; unpublished data). We conclude that germ cells move from the mitotic region into the meiotic zones and that germ cells move through the meiotic region at a rate of ~1 row per hour.
Figure 6. Germ cells move proximally from mitotic region into meiotic zone. Animals were pulsed for 30 min with BrdU and then chased for increasing times. (A–C) Projected z-series of extruded germlines stained with anti-BrdU (pink) and TO-PRO-3 (blue). (more ...)
Movement appeared to stall at a position of ~30 cell diameters from the DTC; that position corresponds roughly to the border between transition zone and pachytene region. The germ nuclei become organized at the periphery of the germline tube when they enter pachytene; perhaps the slowed movement reflects the time it takes for this organization.
We next estimated the number of germ cells in premeiotic S-phase. That estimate is based on the idea that germ cells in premeiotic S-phase will move proximally into the meiotic region and remain intensely BrdU labeled. By contrast, germ cells in mitotic S-phase will divide and label will be diluted during the subsequent S-phase. We found ~60 strongly stained nuclei in the meiotic region after a 30-min pulse and a 16-h chase (n = 11; average 59, range 42–78). With no chase, ~10 nuclei in the transition zone incorporate BrdU, which leaves ~50 nuclei that are likely to have been in premeiotic S-phase within the mitotic region, probably in the 5 to 6 rows just distal to the MR/TZ boundary.
Extent of DTC and Its Processes
Figure 7. Configuration and extent of DTC processes. (A) Adult hermaphrodite germ line, 24 h past mid-L4. Each part of the panel is a projection of two confocal sections in adjacent focal planes from the same germ line. Green, GFP; red, anti-GLP-1; blue, TO-PRO-3. (more ...)
We examined individual confocal sections and followed GFP-positive DTC processes that partially surround or extend between the distal-most germ cells (A). Such processes embraced germ cells in row 1 (17/17 germ lines) as well as cells in rows 2–4 (15/17 germ lines; A). We estimate that ~5 germ cells occupy row 1, ~7 reside in row 2, and ~10 reside in each of rows 3 and 4. Therefore, the DTC processes partially enclose ~30 germ cells in rows 1–4. We suggest that the germline stem cells reside in these distal-most rows of the mitotic region (see Discussion).
We also examined projections of confocal z-series to determine the length of DTC processes along the distal-proximal axis (, B–G). These processes lengthened with age (, H and I). Similar results were found using either transcriptional or translational reporters (, G and J; unpublished data). In contrast to DTC process lengthening, the boundary between the mitotic region and transition zone shortened with age (H). Therefore, DTC process length does not correlate with extent of the mitotic region along the distal-proximal axis (H). Note that although the number of cell diameters between the DTC and TZ decreases with age, the total number of germ cells in the mitotic region remains relatively constant, because of an increased density of nuclei (unpublished data).
We also examined DTC processes in mutants with mitotic regions that are either longer or shorter than normal: DTC process lengths in the mutant were similar to those in wild type, even though mitotic region lengths were different (, E, F, and J). We conclude that the DTC retains extensive contact with the distal-most 3 or 4 rows of germ cells throughout germline development and that DTC process length does not control the length of the mitotic region.
Mitotic Spindles Orient Randomly with Respect to the Distal-Proximal Axis
In the Drosophila
ovary and testis, stem cells reproducibly orient their mitotic spindles so that the self-renewing daughter is born adjacent to the niche, whereas the differentiating daughter is born away from the niche (Xie and Spradling, 2000
; Yamashita et al., 2003
). To ask whether a similar orientation could be observed in the C. elegans
germline mitotic region, we examined the orientation of mitotic spindles, metaphase plates, and/or anaphase chromosomes with respect to the distal-proximal axis of extruded germ lines stained with anti-tubulin and/or DAPI. The plane of division was scored as parallel, perpendicular, or oblique to the distal-proximal axis at each position (A). We found no dramatic bias in orientation at any position (A). In particular, germ cells in the distal-most rows were not reproducibly oriented along the axis (, B–E).
Figure 8. Orientation of cell divisions in mitotic region. (A) The orientation of mitotic spindles and/or metaphase plates was scored as along, across or oblique to the distal-proximal axis at each position. The majority of mitotic germ cells are found in the first (more ...)