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Like many stem cell systems, the C. elegans germ line contains a self-renewing germ cell population that is maintained by a niche. Although the exact cellular mechanism for self-renewal is not yet known, three recent studies shed considerable light on the cell-cycle behavior of germ cells, including a support for significant and plastic renewal potential. This review brings together the results of the three recent cell-based studies, places them in the context of previous work, and discusses future perspectives for the field.
The C. elegans germ line is a strong model for stem cell biology and is poised to become even more powerful. Several features of the C. elegans germ line are analogous to mammalian adult stem cell systems. The germ line is the only C. elegans lineage that proliferates for the life of the organism (Hirsh et al., 1976). The maintenance of germ cell proliferation is governed by interaction with a niche, the distal tip cell (DTC; Kimble and White, 1981), while additional cell-cell communication contributes to the extent of germline proliferation (McCarter et al., 1997; Pepper et al., 2003; Killian and Hubbard, 2005). Finally, like mammalian stem cells, the C. elegans germ line appears to be quite plastic and responsive to changing molecular and physiological conditions. Given that there are several different context-specific paradigms for renewal and maintenance of mammalian tissues, it is necessary to study many stem cell systems to ultimately understand the range of mechanistic paradigms for renewal and its molecular control. Key features of the C. elegans germ line distinguish it from other stem cell models such as the Drosophila germ line, suggesting that it will provide further valuable insights into stem cell biology.
The overall layout of the C. elegans gonad is similar to that of other organisms. Like Drosophila, it is organized as a blind-ended tube with proliferating germ cells at the closed end (distal, in C. elegans) and gametes at the open end (proximal), with the intervening stages of germ cell differentiation in between (Figure 1). In male mammals, the analogous organization axis is from the basement membrane to the lumen of the testes tubule. In all of these organisms, somatic cells are in close contact with proliferating germ cells and regulate their behavior. In C. elegans, the distal tip cell (DTC) acts as a niche that maintains germline proliferation (Kimble and White, 1981). In Drosophila, the interaction between the niche and stem cells is well characterized: contact with the niche defines the orientation of germline stem cell divisions and hence the stem or non-stem fate of the cells (see Fuller and Spradling, 2007). Niche cells are a source of signaling molecules that interact with one or more signal transduction pathways in the germ cells. Although the precise roles of various signal pathways is not conserved in different stem cell systems, the same host of developmental signaling pathways have been implicated in mammalian niche-stem cell interactions as in Drosophila and C. elegans (see Ohlstein et al., 2004; Li and Xie, 2005; Yamashita et al., 2005; Moore and Lemischka, 2006; Naveiras and Daley, 2006; Fuller and Spradling, 2007).
The cellular behavior of stem cells has been determined in several systems. In Drosophila, a series of elegant cell lineage studies have established the existence of “classic” asymmetric stem cell divisions that give rise to two daughters, one of which retains stem cell position, identity and fate, and another that embarks on the path toward differentiation. These stem cell progeny divide in the context of a cyst containing a cohort of lineally related germ cells and encapsulating somatic cells that themselves derive from somatic stem cells (Fuller and Spradling, 2007). A detailed analysis of the behavior of germ cells is aided by markers for stem versus non-stem cells as well as technology to trace lineally related cells. These tools were a prerequisite for understanding the system at the level of individual cells. The study of molecules and interactions that govern Drosophila stem cells has also been taken to the level of individual cells because of these technologies. In mammals, recent advances in technology have also permitted in vivo studies of the male germ cell lineage from stem cells (Nakagawa et al., 2007).
Unlike Drosophila, there is no cyst system in the C. elegans germ line, and germ cells appear to continue on their developmental path en masse (Hirsh et al., 1976). Thus, the study of this system may reveal new insights into the behavior of self-renewing cell populations. The region of the gonad closest to the DTC contains a population of germ cells in various stages of the mitotic cell cycle. Recent data suggest that the germ cell pool is renewed (see below), but because lineage studies have not been conducted to date, the exact cellular mechanism of renewal is unknown. Given the genetic and molecular tractability of C. elegans, it is reasonable to expect that once this system is better defined at the level of the behavior of individual cells, the C. elegans germ line will offer salient insights into the molecular control of stem cells.
The molecular analysis of genes that affect germline proliferation in C. elegans has outpaced a rigorous anatomical analysis of germ cell behavior, leaving a gap in our ability to effectively correlate molecular and cellular aspects of germline proliferation. Several recent studies have addressed this gap, but many critical questions are unresolved. Excellent recent reviews have focused on the molecular aspects of C. elegans germline biology and proliferation (Kimble and Crittenden, 2005; Ellis and Schedl, 2006; Hansen and Schedl, 2006; Kimble and Crittenden, 2007). Therefore, in this article, I will only briefly outline the relevant molecular aspects and will focus instead on the behavior of the proliferating germ cells since they include presumptive stem cells. Many open questions in the field will be presented in the context of three parts. The first part will address relevant anatomical, cellular, developmental and molecular features of the system, focusing on the niche and the behavior of proliferating germ cells in the adult hermaphrodite. The second part will focus on three recent studies that used three distinct experimental approaches to address cell behavior, including cell cycle. The third part will present an emerging view of C. elegans germline stem cells and re-cap the more urgent questions that, once resolved, will enable us to exploit the full potential of this highly tractable model stem cell system.
Before presenting additional information about the C. elegans germ line, two terms relevant to germline stem cells in C. elegans require additional clarification: the term germ “cell” and the terms “mitotic”, “proliferation” or “proliferative” zone. First, the germ line is actually a syncytium. By convention, the term “germ cell” refers to a germ cell nucleus surrounded by its cytoplasm and a cell membrane that retains an opening to a central core of germline cytoplasm called the rachis (Hirsh et al., 1976; Figure 2A, C). A helpful visual analogy is a corncob with the germ cells as large kernels, and the rachis as the core. Although the germ line is syncytial, it is thought that communication between “germ cells” is restricted. For example, neighboring nuclei do not undergo simultaneous cell divisions as would be expected if they extensively shared a common cytoplasm. The absence of truly individualized cells makes it extremely difficult, if not impossible, to perform cell transplantation studies in C. elegans of the kind that have been used to rigorously define stem cells in other systems.
Second, the “mitotic”, “proliferation”, or “proliferative” zone (or region) actually contains at least two different germ cell populations: (1) cells in a mitotic cell cycle and (2) cells that have entered meiotic S-phase (also previously referred to as pre-meiotic S; see Jaramillo-Lambert et al., (2007)). The zone has been defined as the region where mitotic figures can be found, corresponding to the area distal to distinctly crescent-shaped nuclei characteristic of early meiotic prophase (leptotene and zygotene) that mark the start of the “transition zone”. As shown in Figure 1, the different nuclear morphologies in these regions are easily distinguished in a DAPI-stained preparation. Cells that have entered meiotic S phase cannot be distinguished from cells in mitotic interphase using available markers. Cells that have begun meiotic S phase never re-enter mitosis under normal conditions; these cells are neither “mitotic” nor “proliferative”. Therefore, though no single term easily captures the essence of the biology, the term “proliferative zone” may be the least confusing (Francis et al., 1995). An accepted approximation in the field (further supported by studies discussed below) is that cells in the distal half of the adult proliferative zone are in the mitotic cell cycle and that an increasing proportion of cells in the proximal part of the proliferative zone are actually in meiotic S phase. It is also important to note that on average, active cell divisions in the proliferative zone of the C. elegans adult are relatively rare (see below), making it difficult to follow them in real time. Though the position of the distal transition zone border varies somewhat from animal to animal, individual nuclei in the transition zone are virtually always either in meiotic S phase or in early prophase of meiosis I (see below).
Findings of Hansen et al. (2004a) confirmed that immunohistochemical markers for nuclei in the proliferative zone (anti-REC-8 antibodies, under certain fixation conditions) and for nuclei in meiotic prophase in the transition zone (anti-HIM-3 antibodies) label nuclei in the proliferative zone (including those in meiotic S phase) and the crescent-shaped leptotene/zygotene nuclei in the transition zone, respectively, as defined by previous morphological criteria (Hirsh et al., 1976; Hansen et al., 2004a). REC-8 is related to the yeast meiotic cohesion protein Rec8p and, although it localizes to both mitotic and meiotic germline nuclei in C. elegans (Pasierbek et al., 2001), it marks only proliferative zone nuclei under certain fixation conditions (Hansen et al., 2004a). HIM-3 is a meiosis-specific component of the chromosome core that is related to the yeast Hop1p protein (Zetka et al., 1999).
The niche-like relationship between the C. elegans DTC and the germ line, i.e., its role in maintaining germ cell proliferation, was determined by cell ablation studies (Kimble and White, 1981). A single DTC caps the blind end of each gonad arm in the hermaphrodite and a pair of DTCs cap the single gonad arm in the male. Laser-killing of the DTC causes all proliferating germ cells to differentiate. The only signaling pathway identified to date that directly controls the proliferation (versus differentiation) decision by way of DTC-germline interaction is the GLP-1/Notch pathway. Loss-of-function mutations in the genes encoding core components of this pathway largely phenocopy laser-killing the DTC in the sense that all germ cells enter meiosis (with mutations resulting in a slightly earlier defect than cell ablations; Kimble and White, 1981; Austin and Kimble, 1987; Pepper et al., 2003). The ablation studies also established that anatomical mis-location of the DTC can cause ectopic germline proliferation (Kimble and White, 1981). This notion has been further supported by the analysis of certain anatomically restricted germline tumors. These tumors are caused by inappropriate contact between mitotic germ cells and somatic cells, some of which express additional Notch ligands (Seydoux et al., 1990; Pepper et al., 2003; Killian and Hubbard, 2005; McGovern, Maciejowski, Voutev and Hubbard, in preparation). Thus, this relatively simple anatomical system can be exploited to investigate not only the normal niche, but also the effects of various molecular and anatomical alterations that result in a putative ectopic stem cell population maintained by an ectopic niche.
How, exactly, does GLP-1/Notch signaling in the germ line promote proliferation and/or inhibit differentiation? The short answer is that there is, as yet, no clear molecular link between GLP-1/Notch-mediated signal transduction and the control of cell cycle. Briefly, the GLP-1/Notch receptor is activated by the DSL family ligand LAG-2, which is expressed in the DTC. GLP-1 acts in the germ line via a canonical Notch signaling pathway. By analogy from other systems, the intracellular domain of activated GLP-1 is thought to be cleaved upon ligand activation and enter the nucleus where it forms a transcription regulatory complex with a CSL family protein LAG-1 and the SEL-8/LAG-3 protein (see Kimble and Crittenden, 2005; Hansen and Schedl, 2006; Kimble and Crittenden, 2007; and references therein). However, few direct transcriptional targets of this complex have been characterized, nor are molecular markers available as reporters for GLP-1/Notch activity. The identification of additional direct targets of activated GLP-1 is, therefore, a high priority.
Many studies have contributed to the current genetic and molecular understanding of the control of the proliferation/differentiation decision, and the reader is directed to recent reviews for a more thorough discussion and for additional references (Kimble and Crittenden, 2005; Hansen and Schedl, 2006; Kimble and Crittenden, 2007). The most headway has been made in the analysis of germline-autonomous pathways that lie downstream of GLP-1-mediated signaling to interfere with meiotic entry. The story is complex for many reasons, not least of which is that most of these genes encode proteins connected to RNA control, rather than the transcription factors that lie downstream of Notch in other contexts. In short, GLP-1-mediated signaling opposes the activities of at least three partially redundant genetic pathways that lead to meiotic entry and are important to establish the distance to the meiotic prophase (transition zone) border with respect to the distal tip of the gonad (Figure 3; Kadyk and Kimble, 1998; Hansen et al., 2004a, b). Two of these redundant pathways are defined by the activities of GLD-1, a KH-domain containing RNA-binding protein (Francis et al., 1995) and the GLD-2 cytoplasmic poly-A polymerase protein (Wang et al., 2002) that acts in a complex with GLD-3, a Bicaudal C-related protein (Eckmann et al., 2002; Eckmann et al., 2004). The GLD-1 and GLD-2 pathways are not only redundant for meiotic entry, they also interact, forming a complex genetic regulatory network. The data support a general model whereby the outcome of the activated GLP-1 pathway interferes with the accumulation of GLD-1 protein thereby preventing meiotic entry in the distal part of the zone (Hansen et al. 2004b). The pumilio-related proteins FBF-1 and FBF-2 are required redundantly for late larval and adult maintenance of the proliferative zone and mediate the effect of the GLP-1 pathway on GLD-1-dependent meiotic entry (Crittenden et al., 2002; Lamont et al., 2004). The two fbf genes encode very similar proteins and have redundant functions, but they also have some distinct effects on germline pattern. They appear to negatively regulate each other. LAG-1 binding sites are present in the fbf-2 upstream region, suggesting a direct role for FBF-2 in mediating GLP-1 activity (Crittenden et al., 2002; Lamont et al., 2004). Genetic evidence suggests that at least one additional pathway is required to prevent meiotic entry in response to GLP-1 (Hansen et al., 2004a). Several additional genes have been tied to these two pathways, including the nanos-related gene nos-3 that acts upstream of gld-1 for meiotic entry (Hansen et al. 2004b); and atx-2, an ataxin-2 ortholog that promotes germline proliferation downstream of gld-1 and gld-2 or in parallel with GLP-1 signaling (Maine et al., 2004; Ciosk et al., 2004). Interestingly, the fbfs, nos-3, gld-1, gld-2 and gld-3 are all also implicated in germline sex determination, suggesting that control of proliferation and sex determination may be linked (see Ellis and Schedl, 2006; Kimble and Crittenden, 2007). The further elucidation of these pathways and identification of additional direct links between their components and GLP-1 signaling will undoubtedly lead to a deeper molecular understanding of this process in the future.
Another important question is whether the activity of the GLP-1 pathway is transduced to cells far from the DTC. And, if so, how? In the wild type, active germ cell divisions can still be observed ~20–22 “cell diameters” (CD) distance from the distal tip (Figure 1). Each “cell diameter” distance represents the space occupied by a ring of cells that line the periphery of the gonad, and this distance measure has been used in many studies that focus on the control of the size of the proliferative zone. One idea, supported by the observation of a LAG-2 translational reporter fusion in the germ line (Henderson et al., 1994), is that the ligand is taken up by the germ line, perhaps as a late step in receptor endocytosis, and that it continues to signal as the germ cell moves proximally. Cytoplasmic processes that extend from the DTC have also been postulated to deliver ligand to more proximal cells (Figure 2). This idea was supported by the observation of a rough correlation between the length of these processes and the maximum distance at which mitotic figures are observed (Fitzgerald and Greenwald, 1995; Hall et al., 1999). More recent observations, however, suggest that the length of DTC cytoplasmic processes do not always correlate well with the length of the proliferative zone. Crittenden et al. (2006) reexamined the length of DTC processes in younger and older adults and found that the average distance (in CDs) from the distal tip to the transition zone border shortens while the longest DTC processes lengthen as animals age. Thus, the length of DTC processes does not appear to correlate with the mitosis/meiosis border as animals age.
Additional phenomena that may be associated with GLP-1 signal maintenance and/or propagation are thresholds and feedback. The hypothesis that a steep threshold of glp-1 activity governs cell fate is supported by the observation that partial loss-of-function alleles of glp-1 can display an all-or-none phenotype. Under conditions when overall penetrance of the mutant phenotype is low, an individual animal may have a Glp-1 phenotype (few germ cells, all sperm) in one gonad arm and a proliferative germ line in the other gonad arm (Austin and Kimble, 1987; Kodoyianni et al., 1992). One way a steep threshold can be set up and maintained is by a positive feedback mechanism that amplifies initially small differences over a small concentration gradient. Berry et al. (1997) proposed that positive feedback may exist between mitosis and glp-1 activity as supported by their analysis of gain-of-function alleles of glp-1 (Berry et al., 1997). Additional support for this idea comes from the observations that certain partial loss-of-function alleles of glp-1 display a time-dependent shortening of the proliferative zone from proximal to distal (Michaelson and Hubbard, unpublished observations). In addition, the pattern of meiotic entry that follows a shift of conditional alleles to the restrictive temperature occurs in a proximal to distal “wave” of meiotic entry (Hubbard, unpublished observations). Thus, it appears as if proximal cells enter meiosis before more distal cells can do so.
In cellular contexts like the C. elegans germ line, in which one outcome of a Notch-controlled cell fate decision is a “mitotic” or undifferentiated fate, and in which elevated levels of Notch activity are also associated with elevated mitotic index (Maciejowski et al., 2006), there are likely multiple inter-related outputs for the Notch signaling pathway. Notch activity has been implicated in vertebrate cell systems where there is also a context-dependent interplay between Notch signaling as a binary fate switch (e.g., that influences whether a cell adopts or maintains a “mitotic” cell identity versus an alternate differentiated non-dividing cell fate) and as a proliferation-promoting factor in the sense that it affects cell-cycle dynamics (see Maillard et al., 2005; Roy et al., 2007). The anatomical simplicity of the C. elegans germ line and the genetic and molecular tractability of this system offer the potential to tease apart the role of Notch signaling in these processes.
In the C. elegans germ line the GLP-1 signaling pathway promotes a “mitotic” or undifferentiated cell fate and/or inhibiting “meiotic” or differentiated cell fate and may also promote or control cell cycle among mitotic cells thereby affecting the extent of germline proliferation (Figure 3). However, it should be noted that the activity of glp-1 is completely dispensable for proliferation in certain genetic backgrounds such as the gld-1 gld-2; glp-1 triple mutant. Results suggest that cell fate specification and mitosis-promoting functions are related but genetically separable. For example, mutants have been described in which germ cells fail to proliferate properly but do not enter meiosis, even in the absence of glp-1 activity (e.g., glp-4; Beanan and Strome, 1992). Therefore reducing proliferation does not automatically drive germ cells into differentiation. An alternative explanation is that such mutations affect aspects of cell cycle control that are common to mitosis and meiosis causing cells to arrest in an intermediate state. Other mutations and non-DTC cell ablations can reduce germline proliferation even when all cells are in the mitotic cell cycle, as driven by hyper-active GLP-1 function (Hubbard et al., 1996; McCarter et al., 1997; Killian and Hubbard, 2005). These results indicate that optimal germline proliferation requires additional inputs that act in conjunction with GLP-1 signaling (Figure 3). Therefore, there is much remaining to be discovered regarding the contributions of GLP-1/Notch signaling to these two aspects of the control of proliferation and differentiation.
In mammals, the Notch pathway has been directly tied to the cell cycle in different contexts. For example, Notch1 activation in fibroblast and in T-cell acute lymphoblastic leukemia (T-ALL) cell lines promotes down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1) via transcriptional activation of the gene encoding SKP2, the F-box protein subunit of the SCFSKP2 ubiquitin protein ligase complex that targets phosphorylated p27, and thereby accelerates the G1 transition (Sarmento et al., 2005; Dohda et al., 2007). The results of Maciejowski et al. (2006) in C. elegans suggest that GLP-1/Notch activity has a greater impact on the cell cycle of putative transit-amplifying cells than putative stem cells (see below), but direct cell-cycle connections remain to be elucidated. If Notch affects transit amplification more than stem cell divisions in mammals, there would be important implications for cancer therapies that target Notch signaling.
In mammalian stem cell systems, Notch-mediated signals also act in concert with other signal transduction pathways, such as the Wnt, Hedgehog and Insulin/IGF pathways, and it is in the context of interaction with these other pathways that the final phenotypic consequences of receptor activity are observed. As a result of these interactions, Notch can act both as a tumor promoter and a tumor suppressor, depending on context (see Roy et al. (2007) and references therein). While no C. elegans genes other than those involved in GLP-1 signaling have yet been found to have the same dramatic effect on the mitosis/meiosis decision, it is becoming apparent that other signal transduction pathways collaborate with the GLP-1/Notch pathway in the C. elegans germ line and influence the extent of proliferation both under normal conditions and nutrient-restricted conditions (Fukuyama et al., 2006; Narbonne and Roy, 2006; Michaelson and Hubbard, unpublished observations). In one case, an unexpectedly direct link was uncovered between the MAP kinase pathway and GLP-1 signaling (Lee et al., 2006). The initial observation was that a mutation in the lip-1 gene, which encodes a predicted map kinase phosphatase, reduced the number of cells in the adult germline proliferative zone. In addition, the lip-1 promoter immunoprecipitates with a SEL-8/LAG-3 peptide and its mRNA appears to be directly regulated by FBF (Lee et al., 2006). The intersection of signaling pathways in the control of stem cell biology is a very exciting area where a relatively simple genetically accessible organism like C. elegans will likely continue to provide important insights since the effects of these interactions can be manipulated and characterized in vivo.
The proliferative zone is not a steady-state cell population in terms of cell number, nor in terms of physical volume of the gonad that it occupies over the course of development. Most studies have focused on the adult hermaphrodite mitotic zone. In larval stages, however, the mitotic zone comprises fewer cells. About 50 germ cells (13 CD from the distal tip) occupy each hermaphrodite gonad arm mid-way through the third larval stage (L3) when the first evidence of meiotic prophase can be seen. Prior to the mid-L3, all the germ cells are “mitotic” in the sense that they have not entered prophase of meiosis. However, they must first enter meiotic S. Once the DTC is far enough away from more proximal germ cells, the proximal-most germ cells enter meiotic prophase (“initial meiosis”; Pepper et al., 2003). An important finding is that the border between “mitotic” and “transition” nuclei in the L3 and up to the mid-L4 stage is abrupt and occurs over a distance of one cell-diameter (approximately 4 μm) (Hansen et al., 2004a). That is, in wild-type larvae at the time of initial meiosis, all cells within the distal 13 cell-diameters distance from the distal tip display nuclear morphology and antibody-labeling consistent the proliferative zone, while all cells in the next cell-diameter (or next ring of cells) display nuclear morphology and antibody-labeling consistent with prophase of meiosis I. Thus, initial meiosis establishes the germline pattern by restricting proliferating germ cells to the distal part of the gonad arm and defining the size of the potential stem cell pool. Over the course of the L4 and early adult stages, simultaneous with the continued meiotic entry and meiotic progression of the proximal-most cells, the pool of proliferative zone cells increases in number to about ~250 cells occupying the distal-most ~20–22 CD from the distal tip in the early adult.
The ability of the gonad arm to attain the critical distance from the DTC and thereby permit initial meiosis (13 cell-diameter distance in wild-type larvae), is a function of two independent processes: (1) gonad migration led by the DTC (Kimble and White, 1981) and (2) the space-filling capacity of the germ cells, which is directly related to the number of the germ cells. The gonad is somewhat elastic; a useful visual analogy is an empty balloon versus a full balloon. As a result of this elasticity, the DTC of a gonad arm containing fewer germ cells does not extend as far from the proximal germ cells and the central gonad primordium as does the DTC of a gonad arm containing the proper complement of germ cells. Thus, in certain mutants in which early germline proliferation occurs but is reduced, delayed and/or inefficient, the DTC remains too close to the proximal-most germ cells in L3 larvae and hence the switch from the mitotic cell cycle to meiotic entry among proximal germ cells is delayed (Killian and Hubbard, 2005; Voutev et al., 2006). Under these circumstances, initial meiotic entry can be delayed relative to the development of the somatic cells of the gonad. This situation – delayed or retarded germline differentiation in the context of a more normally-timed somatic gonad development – can, in turn, set up inappropriate cell-cell interactions that result in the formation of a proximal germline tumor (Killian and Hubbard, 2005). Similarly, in mutants in which DTC migration is delayed early in larval development, meiotic entry is also delayed (Tamai and Nishiwaki, 2007; Voutev and Hubbard, unpublished observations). These effects must all be taken into account when considering the dynamic events that influence the establishment of the pool of mitotic germ cells in which the putative stem cells reside.
In contrast to the sharp proliferative zone/transition zone border in the L3 and into the L4, the late-L4 and adult distal mitotic zone contains an increasing number of cells in a staggered sub-region at the border of the mitotic zone and transition zone that contains both mitotic and transition nuclei. This overlap has been dubbed the “meiotic entry region” (Hansen et al., 2004a; Figure 4). The staggered nature of this border led to slight discrepancies in the literature regarding the criteria by which the position of the proliferative zone/transition zone border was delineated at the single-cell level. The definition used in one of the recent studies (Crittenden et al., 2006) captures the essence of the biology: the distal border of the transition zone is defined as the first ring (CD) of nuclei with more than one transition nucleus. When the pattern of germ cell development is normal, more than one crescent-shaped or HIM-3-positive nucleus is virtually always observed in the first row of germ cells that contains these nuclei (Figure 1). When the pattern of germ cell progression from mitosis to meiosis is disrupted, however, the use of antibody markers is preferred to define the state of individual cells (Hansen et al., 2004a).
Given that the entire cell lineage in C. elegans has been followed from zygote to adult, why is there any question concerning the existence, identity, location and cell division behavior of putative germline stem cells? The answer is that in contrast to the predominantly invariant and fully described cell lineage of the C. elegans soma (Sulston and Horvitz, 1977; Kimble and Hirsh, 1979; Sulston et al.,1983), germ cell divisions are variable with respect to order and division plane (Kimble and Hirsh, 1979). Thus, the complete cell lineage of C. elegans that has permitted unprecedented single-cell resolution correlation of genetic and anatomical studies in this organism is not applicable to the germ line. In this regard, the germ line is more akin to other systems where cell lineage within a dividing tissue is variable from individual to individual (see below for a discussion of cell lineage approaches for the germ line). Moreover, no molecular markers have yet been characterized that can distinguish cells from the mitotic cell cycle from those in meiotic S phase; nor do any molecular markers exist for germline stem cells.
Despite the lack of markers for germline stem cells, their existence has been postulated largely on the reproductive capacity of the worms. Early studies of C. elegans gonadogenesis suggested a very fast (6.5 hours) rate of turnover of the gonad volume (Hirsh et al., 1976). The C. elegans hermaphrodite gonad produces approximately 300 sperm before switching to oogenesis after the adult molt. Oocytes mature and are ovulated into the spermatheca one at a time where they are fertilized by sperm (either self-sperm or male sperm from mating). Continuous mating can produce ~1400 progeny from a single hermaphrodite (Kimble and Ward, 1988). Therefore, the question of germ cell renewal becomes: how many germ cells can become oocytes? Based on the ~200–250 germ cells in the adult proliferative zone, two diametrically opposed models are as follows. (1) There is no renewal mechanism per se and all mitotic germ cells undergo 2–3 divisions over time and go on to form the necessary complement of germ cells and gametes. (2) There is a renewal mechanism that maintains a mitotic population of cells; these cells can leave the mitotic cell cycle by entering meiotic prophase, and it is these additional cells that support the continuous production of gametes. There is now firm support for the renewal mechanism, as indicated below.
Three recent studies have addressed questions related to cell cycle behavior of germ cells using different approaches. First, I summarize the approaches and then synthesize the relevant results of these studies that impact our understanding of germ cell development and the behavior of putative germline stem cells.
Maciejowski et al. (2006) examined the frequency of active mitosis (metaphase and anaphase) as a function of distance from the DTC in the wild type and in a mutant with a hyperactive Notch receptor that exhibits an enlarged proliferative zone. Starting with a relatively small data set (n=50) and a manual analysis of mitotic index, this study turned to computer-assisted data collection on a larger data set (n>500) and statistical analyses to establish rigorous benchmarks and to circumvent problems associated with the relatively low frequency of active cell divisions. In addition, the analysis examined the occurrence of simultaneous divisions and their spatial clustering.
Crittenden et al. (2006) addressed the question of germ cell renewal and applied traditional BrdU feeding/labeling techniques to explore germline cell-cycle dynamics. Extending previous analyses and using methods that labeled the DTC, this study also investigated the anatomical relationship of the distal tip cell (cell body and its cytoplasmic processes) to underlying germ cells and to the border of the proliferative and transition zones. Finally, the orientation of germ cell division axis with respect to the distal-proximal gonad axis was investigated.
Jaramillo-Lambert et al. (2007) monitored S phase (both mitotic and meiotic) by introducing fluorescently labeled nucleotides directly into the germ line and monitoring their incorporation. This approach was used under a wide variety of conditions to define the pattern of label up-take, the relative timing of mitotic and meiotic S phase, chromosome-level patterns of S phase completion, the timing of meiotic progression and its response to sexual identity and to the presence of sperm, and the extent of germ cell death during oogenesis.
As the adult animal ages and gametes are used, germ cells must continue to divide if the number of cells in the proliferative zone is to remain constant. However, there are conflicting reports as to whether the proliferative zone cell numbers are maintained over time as adults age. Data presented by Crittenden et al. (2006) indicate that the number of germ cells in the proliferative zone is relatively constant as animals over a week of adulthood (an average of 243 cells on day 1 and 238 on day 6). Crittenden et al. note a decrease in the size of the zone over adulthood (as measured by CD distance from the distal tip), citing unpublished data indicating that the density of the germ cells increases, thereby supporting their observation of constant cell numbers despite the apparent shortening of the zone. In contrast, Killian and Hubbard (2005) reported a decrease in the number of germ cells in the proliferative zone as animals aged over two days: a loss of over 35% (an average of 251 to 156 cells). It is possible that the strain used by Killian and Hubbard, which contained two integrated transgene markers, influenced the germline response to aging. Indeed, this strain is slow-growing compared to the wild type, reaching adulthood ~10 hours later than the wild type. We recently re-examined a laboratory stock of wild-type worms (N2 strain), and observed a more modest decrease in germ cell numbers, but a decrease nonetheless. Over a 3 day period, the number of proliferative zone nuclei went from 207±14 cells (±SEM, n=15 gonad arms) in the early adult to 179±18 (n=11) cells, a decrease of 13% from the peak number of germ cells (p <0.05; Michaelson and Hubbard, unpublished data).
These conflicting results suggest that the maintenance or renewal capacity of the germline proliferative zone over time may be sensitive to particular laboratory conditions (media, bacteria, etc.) and/or to genetic differences. The former suggests an environmental component to maintenance of the proliferating germ cell pool, while the latter suggests a genetic basis for maintenance of the zone as a function of age. Both of these possibilities point to interesting avenues for further investigation of the influence of aging on the maintenance of the germ line, and justify additional study.
Regardless of the degree to which the proliferative zone is maintained over time in the adult, the idea that significant renewal must occur during the peak reproductive period is strongly supported by recent studies. Results of pulse-chase experiments reported by Crittenden et al. (2006) establish that all germ cells in the zone are actively cycling, while data from Jaramillo-Lambert et al. (2007) provide a new measure for the number of cells that begin meiotic prophase and subsequently undergo programmed cell death. It appears that a far greater number of germ cells undergo programmed cell death than was previously thought. During oogenesis, some germ cells that begin prophase of meiosis I, proceed to the pachytene stage, and then undergo programmed cell death. These cells likely donate their contents to cells that will complete oogenesis and thereby act as nurse cells (Gumienny et al., 1999). Initial estimates were that for each cell that became an oocyte at least one germ cell died (Gumienny et al., 1999). Jaramillo-Lambert et al. followed labeled cells as they traversed prophase of meiosis and found that as many as 30 cells were lost for each cell that formed an oocyte: from 100 nuclei that were labeled in pachytene, ~3 were still labeled in diplotene/diakenesis. When these cell deaths are prevented by a mutation in ced-4, the number of germ cells that were followed remained constant over the course of the experiment, indicating that the loss was due to programmed cell death.
Given the rate of oocyte maturation (McCarter et al., 1999) and the new cell death estimate from Jaramillo-Lambert et al., a rough calculation can be made regarding the required number of doublings that must occur in the proliferative zone to account for germ cell loss to programmed cell death and oogenesis. An adult hermaphrodite with a reserve of sperm ovulates approximately once every 23 minutes per gonad arm (McCarter et al., 1999), thereby producing ~60 oocytes in 24 hours. Based on the intensity of BrdU labeling, Crittenden et al. (2006) estimate that ~50 cells in the proliferative zone are in meiotic S (Crittenden et al., 2006), leaving ~200 that are actively dividing. Based on the results of Jaramillo-Lambert et al., ~30 cells undergo programmed cell death for each oocyte formed. Thus, using these estimates, the mitotic cell population must double over 3 times in 24 hours to maintain its numbers (that is, 60 oocytes + (60*30) or 1860 new cells must be produced in 24 hours). This new estimate of the number of germ cell deaths per oocyte formed is an important parameter in estimating the requirements of the C. elegans oogenic germ line for maintenance and renewal. Taken together, the data support the second model, that there is renewal in the germ line.
From the standpoint of germ cell renewal and its potential plasticity, another fascinating aspect of the Jaramillo-Lambert et al. investigation concerns the control of the rate of meiotic progression. The results indicate that the duration of meiotic prophase is quite plastic and can be altered at several different stages in meiotic prophase I. Older adult hermaphrodites and female germ cells progress through meiotic prophase more slowly than younger controls and male germ cells, respectively. The delay in meiotic progression in older hermaphrodites occurs in the leptotene/zygotene stage (transition zone), while female germ cells relative to male germ cells prolong prophase in the pachytene stage. Each male germ cell gives rise to 4 sperm, and germ cells destined to become sperm do not undergo cell death (Gumienny et al., 1999). Jaramillo-Lambert et al. found that in males, progression through meiotic prophase occurs in 20–24 hours rather than the 54–60 hours required for female germ cells, with the duration of pachytene accounting for much of the difference. The rate of oocyte maturation and ovulation in C. elegans is dependent on the presence of sperm, and significant headway has been made in uncovering the molecular basis for this response (see Greenstein, 2005 and references therein). Jaramillo-Lambert et al. found that in hermaphrodites with no sperm, the duration of meiotic prophase was extended by at least 12 hours in the pachytene stage, and the delay was reversed when sperm were reintroduced by mating.
What happens to germ cell proliferation dynamics when germ cells stall in meiotic prophase? It will be of interest to explore further the cell cycle parameters under additional conditions such as germ cells destined to become male and during aging. It will also be of considerable interest to determine whether the rate or pattern of germ cell proliferation is affected when the duration of meiotic prophase changes in hermaphrodites in response to the presence of sperm.
The original designation of the “proliferative” zone indicated the entire zone, distal to meiotic prophase, where active mitosis could be observed, even if rarely. While this is still a useful definition, together with results of Hansen et al. (2004a), all three recent studies featured here contribute to a more clear picture of the cell cycle behavior within the early adult proliferative hermaphrodite zone as a function of distance from the distal tip. Based on these findings, 4 partially overlapping sub-zones can be identified within the proliferative zone: (1) the distal-most zone comprising germ cells in the first 1–2 cell-diameters from the distal tip that are nestled against the DTC cell body, (2) a zone of highest average mitotic index that encompasses the germ cells up to the mid-point of the distance between the DTC and the first row of multiple transition nuclei, (3) a more proximal zone of declining average mitotic index from (~CD 10–16), and (4) a low mitotic index zone (~CD 17–22). The last two zones likely include cells completing their last mitotic cell cycle and cells in meiotic S phase. The transition zone then begins where some nuclei have entered meiotic prophase. Each of these sub-zones will be considered in turn (Figure 1).
The germ cells in the distal-most sub-zone or first few cell diameters from the distal tip are distinguished from their near neighbors in more proximal positions by behavioral and anatomical phenotypes: they display a lower mitotic index (Crittenden et al., 1994; Crittenden et al., 2006; Maciejowski et al., 2006), a less robust up-take of fluorescently labeled dNTPs (Jaramillo-Lambert et al., 2007) and intimate contact with the DTC body (Crittenden et al., 2006; Figures 1, ,22 and and55).
The data also indicate that these cells are not quiescent, since cell divisions are observed among them (Crittenden et al., 2006; Maciejowski et al., 2006; Figure 5). In addition, they can take up BrdU label indicating that they are moving through S phase, and the label can be subsequently chased from them indicating that they are moving out of S phase (Crittenden et al., 2006). Maciejowski et al. measured mitotic index directly by counting the number of actively dividing cells (metaphase or anaphase) and dividing by the average cells per CD in 50 gonad arms. The average mitotic index was 0.7% for cell diameters 1–2 and 1.36% for CD 3–10, indicating that the mitotic index is ~50% lower in the first two cell-diameters. The observation of a lower mitotic index in these cells led to the speculation that they may be similar to other stem cell systems such as the mammalian gut crypt in which the stem cells cycle more slowly than the transit amplifying cells further from the niche (Maciejowski et al., 2006). Crittenden et al. (2006) reported mitotic index as phosphohistone-H3-positive cells (prometaphase and metaphase) at each position in 102 gonad arms and divided by an average number of germ cells obtained from an independent experiment. For the 1–2 CD and 3–10 CD intervals, respectively, the mitotic index was 2.9% and 4.3%, a ~30% difference in the mitotic index for these two regions. Both of these measures are comparable to mammalian stem cell systems. For example, a two-fold difference in cell cycle time is estimated between stem cells and their transit amplifying cell progeny in the gut crypt (Potten, 2004). A comparison of the data from these two studies further suggests that the percentage of the cell cycle that is detected with antibody against phospho-histone-H3 is about 25–30% longer than that measured by nuclear morphology (metaphase and anaphase). Since only an average 1% of cells are observed in metaphase/anaphase (Maciejowski et al., 2006), this suggests that the total time of pro-metaphase through anaphase is, at most, an average of ~5% of the total cell cycle time.
Maciejowski et al. (2006) made the additional observation that the expected probability of observing a dividing cell in the distal-most part of the zone did not increase when GLP-1/Notch activity was elevated. The expected probability was elevated in the cells proximal to this DTC-associated region. These observations further support the supposition that the control of the cell cycle in the distal-most zone is different from that of cells somewhat more proximally located. It will be of interest to determine what molecular or anatomical factors influence this difference.
If the reduced mitotic index is a reflection of a slower cell-cycle time in the distal-most cells, one expectation is that the S-phase index (the percentage of cells labeling with BrdU after a short pulse) should also be lower in the distal-most cells. The S-phase index measured by Crittenden et al. (2006), however, does not indicate a statistically significantly lower overall labeling in the distal-most 2 CDs. The combined S-phase and mitotic index observations in this study led to the speculation that the M phase may occupy a shorter relative time of the cell cycle in the distal-most cells (Crittenden et al., 2006). While this possibility cannot be ruled out, the trend in the average S-phase index from the data presented (based on 12 gonad arms) is provocative and suggests that a larger-scale study may be warranted. In addition, Jaramillo-Lambert et al. observed a dearth of distal-most cells incorporating fluorescently labeled dUTP, suggesting that the effective S-phase index may, indeed, be lower in these cells. Another explanation for this result is that the distal cells may be less accessible to the label. However, the authors note that altering the distal-proximal position of injection did not appear to alter the pattern of labeling (Jaramillo-Lambert et al., 2007). It will be important to establish the extent to which cell cycle is delayed and/or which part of the cycle is altered in distal-most cells relative to their neighbors. The significance, if any, of this delay on the stem versus non-stem nature of the distal germ cells will also be important to establish.
An additional tantalizing anatomical feature of the distal-most section of the gonad is that the DTC not only “caps” the distal-most germ cells but seems to envelop the distal-most germ cells more completely than those further proximal (Crittenden et al., 2006; D. Hall, unpublished; Figure 2). This “hugging” of germ cells by the DTC suggests the designation “DTC-associated germ cells”, for the distal-most germ cells. In the adult, the first 1–2 CD contain an average of ~12 cells (Crittenden et al., 2006; Maciejowski et al., 2006). These cells have the largest area of their cell membranes in direct contact with the DTC cell body. Crittenden et al. estimate that ~30 cells within the first 4 CDs of the adult are anchored by the DTC. The observation that these cells are in more intimate contact with the DTC niche cell led to the speculation that germ line stem cells may reside in this section (Crittenden et al., 2006). These speculations are consistent with the model suggested by Maciejowski et al. whereby the proliferative zone behaves as a stem cell/transit amplifying system but remain to be tested by further analysis (see below).
The steady-state levels of several proteins known to affect the position of the proliferative zone border are non-uniform across the proliferative zone including GLP-1, FBF-1, FBF-2, and GLD-1 (Hansen et al., 2004b; Lamont et al., 2004; Crittenden et al., 2006; Figure 5). How, exactly, the measurable steady state levels of GLP-1 correlates to activity of the receptor is not yet known. In the DTC-associated sub-zone, relatively high FBF-2 protein levels are observed while, the level of FBF-1 protein is lower relative to cells further proximal (Lamont et al., 2004). Further studies are needed to understand fully the significance of these protein expression patterns with respect to germ cell behavior.
The next-proximal sub-zone contains cells with the highest average mitotic index and includes germ cells in the next ~7 cell diameters (~CD 3–10; Figure 1). Approximately 90 cells occupy this region (Maciejowski et al., 2006). The average number of cells that readily label with fluorescent dUTPs and with BrdU is uniform across this sub-zone (Crittenden et al., 2006; Jaramillo-Lambert et al., 2007; Figure 5). Together with the mitotic index results, data suggest that S-phase labeled cells in this sub-zone are in the S phase of mitosis (as opposed to meiosis). The steady-state protein expression profile of these cells includes high levels of membrane-bound GLP-1, high levels of FBF-1 and FBF-2, and increasing (though not peak) levels of GLD-1 across the sub-zone. In the absence of GLP-1, the level of GLD-1 protein in this zone is uniformly high, providing evidence that GLP-1 negatively regulates GLD-1 protein levels (Jones et al., 1996; Hansen et al., 2004b). This sub-zone also includes a high level of germline-internalized LAG-2::beta-Gal(intra) protein (Henderson et al., 1994).
The next sub-zone encompasses the cells in the ~10–16 CD range. This area is characterized by an overall decrease in average mitotic index compared to the sub-zone of highest mitotic index, though a reproducible spike or bump in frequency of mitoses is observed (Crittenden et al., 1994; Hansen et al., 2004a; Crittenden et al., 2006; Maciejowski et al., 2006; Figure 5). Based on the estimates of cell cycle from Crittenden et al. (2006) and Jaramillo-Lambert et al. (2007) cells in this zone are likely either in their last mitotic cycle or in meiotic S phase. Taking advantage of available mutants with a high proportion of germ cells in mitosis versus meiosis and vice versa, Jaramillo-Lambert et al. estimate that mitotic S phase is at least two-fold faster than meiotic S phase. Assuming that label can be incorporated throughout S phase, the somewhat elevated average incorporation of fluorescently labeled dUTP in this region observed by Jaramillo-Lambert et al. is likely a result of the increased probability that cells in this region will be in S phase since it occupies a longer time.
Regarding protein levels, the steady-state levels of intensely staining membrane-bound GLP-1 begin to decline over this region (though internal GLP-1 levels remain high; Crittenden et al., 1994), while levels of GLD-1 continue to increase over the zone (Jones et al., 1996; Hansen et al., 2004b).
The sub-zone from CD ~17–22, contains approximately 50 cells in which the probability of observing active mitosis is lowest, and most cells are in meiotic S (Crittenden et al., 2006). This sub-zone is also characterized by decreasing levels of membrane-bound GLP-1, increasing GLD-1, and decreasing FBF-1 and FBF-2 (Crittenden et al., 1994; Jones et al., 1996; Hansen et al., 2004a, b; Lamont et al., 2004).
Data presented by Jaramillo-Lambert et al. (2007) suggest that the region between CD 21 and 30 (that roughly coincides with the “meiotic entry region” (CD 20–29) characterized by overlap of REC-8-positive/HIM-3-negative cells and REC-8-negative/HIM-3-positive cells; Hansen et al., 2004a) contains cells that are exclusively in meiotic S and early meiotic prophase. Thus, under the fixation conditions used by Hansen et al. (2004a) all of the REC-8 positive cells in this region are likely in meiotic S phase.
Interestingly, Jaramillo-Lambert et al. observe Cy-3-dUTP incorporation in nuclei in this sub-zone while Crittenden et al. do not see BrdU labeling in the same region (Figure 5). It is possible that the Cy-3-dUTP method over-estimates cells in S phase while the BrdU labeling underestimates cells in S phase. However, because neither method can distinguish mitotic from meiotic S phase, further investigation will be required to accurately delineate the location of individual cells in meiotic S phase.
The method employed by Jaramillo-Lambert et al. allowed them to make several additional interesting observations. In one experiment, they monitored fluorescent dUTP uptake and the loading of HIM-3, a component of the axial element of the synaptonemal complex onto chromosomes. They found that at the proliferative zone/transition zone border, most nuclei labeled with one or the other marker indicating that meiotic S phase is completed prior to the loading of HIM-3. However, some nuclei labeled with both markers, indicating that there is some temporal overlap of these two processes. Another set of experiments led to the interesting conclusion that the heterochromatic-like X chromosome replicates later than the autosomes during meiotic S in both males and hermaphrodites.
Using independent measures, Crittenden et al. (2006), and Jaramillo-Lambert et al. (2007) estimate the average time of S-phase at 8–12 hours and a maximum of 6 hours, respectively, both relatively long. Crittenden et al. estimate cell cycle time in the first 16 CDs of the adult mitotic zone as 16–24 hours. They note that this estimate contrasts sharply with the ~4 hour doubling time for germ cell numbers in the larval stages (Kipreos et al., 1996). In the early embryo where the cell cycle alternates between M and S without intervening gaps, the cell cycle is about 15 minutes, with M phase (nuclear envelope break-down to cytokinesis) occupying less than 2 minutes (Brauchle et al., 2003). Taken together with the data from Maciejowski et al. (2006), at the lower estimate of a 16 hour cell cycle, these data suggest that metaphase/anaphase would average over 9 minutes and the time of phospho-histoneH3 positive would be on the order of ~30 minutes. The estimated adult germ cell cycle time is also long compared to the post-embryonic somatic cell divisions, which rarely approach 20 hours and are most often less than 10 hours in duration (Sulston and Horvitz, 1977; Kimble and Hirsh, 1979).
This relatively long average cell cycle time in the adult raises additional questions and must be taken into account in further studies. The results of all three studies suggest that an average cell cycle time is, itself, an inadequate estimate for the real dynamics in the germ line, since the mitotic index is different in the sub-zones of the proliferative zone. Moreover, the global changes in cell-cycle time over the larval-to-adult transition must be taken into account in experiments that use real time to trace events in the germ line. It will be of great interest to determine by lineage studies whether the cell-cycle time is similar or different depending on the previous cell division history of a cell. It will also be of interest to determine whether all cells undergo the same number of cell divisions as they traverse the proliferative zone or whether some cells cycle more than others.
In Drosophila, the germline stem cells remain in the niche – either in contact with the terminal filament in females or with the hub in males – while the daughter cell is excluded from the niche by virtue of the cell division plane. Unlike Drosphila, specialized intercellular junctions have not been found between the DTC and neighboring germ cells in C. elegans (Hall et al., 1999; Lints and Hall, 2005; Crittenden et al., 2006). Crittenden et al. (2006) examined the division plane of dividing germ cells by scoring metaphase/anaphase nuclei and/or mitotic spindle orientation and found that the cells were not biased with respect to the distal-proximal axis of the gonad arm. Because of the cap-like shape of the DTC, however, although cell divisions are not oriented with respect to the distal-proximal axis of the gonad arm, it remains to be determined whether their orientation is biased at all with respect to the point of greatest contact with the DTC body or its processes. It is still not known whether one of the two daughter cells more frequently maintains surface contact with the DTC while the other daughter cell eventually moves away from the DTC (either proximally or toward the center). Most importantly, it must be determined whether the fate of a cell that maintains close contact with the DTC after division is different from one that does not.
Once a cell population is found to be self-renewing, the next question is: what is the cellular mechanism of this renewal? Stem cells are defined as cells that can give rise to progeny capable of renewing and progeny capable of differentiating (Watt and Hogan, 2000). This outcome can be achieved by a single stem cell that continually gives rise to one stem-like daughter and one daughter that either differentiates directly or divides further to generate cells that all undergo differentiation. The same outcome can be achieved by a population of cells, including a mixture of cells that can undergo either symmetric divisions that produce two stem cells, asymmetric divisions that produce one stem cell and one differentiated cell, and symmetric divisions that produce two differentiated cells. In this context, the terms symmetric and asymmetric refer to developmental potential and ultimate fate of the daughter cells; differences or similarities in daughter cell sizes may or may not correlate to differences in fate. Provided that the total number of daughter cells with continued renewal potential equals the number of daughters that will only contribute to differentiated cell cohorts, the stem cell population will be maintained. In niche-based systems, the niche can maintain single or small numbers of asymmetrically dividing cells, or a combination of asymmetrically dividing and symmetrically dividing cells, the latter replacing vacancies that might appear in proximity to the niche (Morrison and Kimble, 2006; Fuller and Spradling, 2007).
The exact cellular mechanism of C. elegans germline renewal is unknown. It is plausible that a population-based mechanism is at work, as proposed by Morrison and Kimble (2006). Many questions still remain such as: Does renewal occur on the scale of the entire mitotically active part of the proliferative zone? Or are the DTC-associated cells a stem cell population? That is, do cells located in closest proximity to the DTC – and they alone – produce progeny that are destined to renew and differentiate? Are renewing divisions symmetric or asymmetric or a mixture? Thus one of the open challenges for the field is to determine how cell division history (lineage) correlates with fate in the germ line.
Another plausible mechanism for renewal is that the cell divisions may be neither symmetric nor asymmetric per se, until they experience a low threshold of GLP-1 activity. As proposed by Hansen et al. (2004a), once initiated in the DTC-associated germ cells, the cell cycle may be more or less autonomously regulated. In this scenario, the only relevant factors are (1) the level of GLP-1 activity that cells experience as they move (or, more likely, are pushed) proximally and (2) their place in the cell cycle. For example, cells may only assess the level of GLP-1 activity in a certain part of the cell cycle (e.g., G1) and then decide whether to enter another mitotic S phase or meiotic S phase. An alternate but similar mechanism would be that germ cells assess the level of GLP-1 activity throughout the cell cycle but can only act on the cell fate decision to exit the mitotic cycle in the G1 to S transition.
Several experimental observations are consistent with a cell-cycle-coupled GLP-1 response mechanism. Maciejowski et al. (2006) observed loose spatial and temporal clustering that could be the remnant of lineally related cells dividing in loose synchrony. Preliminary data indicate that “waves” of more tightly synchronized cell divisions – as measured by peaks of mitotic division frequency in fixed animals – are seen in larval germ lines, a result that is consistent with this model and may account for the sharper proliferative zone/transition zone boundary observed in larval germ lines (Hansen et al., 2004a; Maciejowski and Hubbard, unpublished observations). A cell-cycle regulated response to GLP-1 is also consistent with data from at least one other Notch-mediated cell-fate decision in C. elegans, vulval precursor cell fates influenced by LIN-12/Notch (Ambros, 1999).
“Stem cell divisions” and “GLP-1 response” mechanisms for C. elegans germ line renewal are not mutually exclusive. For example, cells in the DTC-associated sub-zone may undergo asymmetric and/or symmetric stem cell divisions, while the putative transit-amplifying cell population assesses GLP-1 activity levels. This combination model is consistent with the observations made by Maciejowski et al. (2006): the relatively low frequency of cell division among DTC-associated germ cells does not change in response to an increase in GLP-1 activity, while more proximally, germ cells are GLP-1-responsive.
To distinguish between these models, it will be necessary to trace the lineage (position, cell division history and fate) of individual germ cells as they traverse the proliferative zone and enter meiosis. These experiments are technically challenging and will require the development of techniques new to C. elegans. One promising technique is an adaptation of the “flp-out” system similar to one used in Drosophila (Gonczy and DiNardo, 1996), that can provide a fate-independent lineage mark. This technique has been developed for C. elegans somatic cells (Voutev and Hubbard, in preparation); it is being further developed for germ cells. In addition, molecular markers that distinguish the DTC-associated cells from their progeny will be extremely valuable. If the DTC-associated cells are stem cells and cells proximal to them are transit amplifying cells, another question is whether, as in Drosophila (Brawley and Matunis, 2004) and mouse (Nakagawa et al., 2007) germ line systems, the transit amplifying cells retain some “stemness” and the potential to re-populate the stem cell niche. This possibility was first postulated for hematopoetic stem cells (Potten and Loeffler, 1990).
Once germ cells enter the sub-zone of high mitotic index they are likely pushed proximally by the divisions of cells distal to them (or perhaps both pushed by divisions and pulled by vacancy), and intercalate with progeny of other DTC-associated cells. Based on calculations made above, on average, the daughters of the DTC-associated cells would divide once they are clear of the DTC body and likely divide a third time before entering meiotic S phase. This model could account for the loose temporal and spatial clustering of M phase nuclei observed by Maciejowski et al. and for the number of doublings required to maintain the zone.
Other important aspects of the proliferating germ cell population in C. elegans require additional experimental attention. More direct measures of cell cycle time – including measures of G1 and G2 – as a function of distance from the distal tip are required. Once determined, the potential changes in cell cycle dynamics (both cycle time and pattern) will be important to examine under conditions that alter meiotic progression and/or reproductive output, such as germ cell sexual identity, age, sperm availability, and environmental conditions such as nutrition. In addition, in order to link the molecular and behavioral aspects of germline proliferation and potential stem cell behavior, it will be important to rigorously determine the effects of specific mutations on the cell cycle.
Already, analogies can be made, both molecular and anatomical, between properties of the C. elegans germ line and mammalian stem cells. Several mammalian stem cell systems appear to be governed by a population-based mechanisms, and in some cases, Notch signaling is involved in their control (see reviews by Ohlstein et al., 2004; Naveiras and Daley, 2006). Further characterization and manipulation of germline stem cells in C. elegans will facilitate additional fruitful comparisons and analogies with the various adult stem cell populations in mammalian tissues.
I gratefully acknowledge David Hall for providing unpublished data. I also thank many colleagues for discussions and Dave Hansen and members of my laboratory for comments on the manuscript. Research in my laboratory is supported by the Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute for Biomolecular Medicine, the Helen and Martin Kimmel Center for Stem Cell Biology, the NIH, and the March of Dimes Birth Defects Foundation.