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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Pathol. Author manuscript; available in PMC 2010 August 27.
Published in final edited form as:
PMCID: PMC2929242
NIHMSID: NIHMS224603

Purpose and regulation of stem cells: a systems-biology view from the Caenorhabditis elegans germ line

Abstract

Stem cells are expected to play a key role in the development and maintenance of organisms, and hold great therapeutic promises. However, a number of questions must be answered to achieve an understanding of stem cells and put them to use. Here I review some of these questions, and how they relate to the model system provided by the Caenorhabditis elegans germ line, which is exceptional in its thorough genetic characterization and experimental accessibility under in vivo conditions. A fundamental question is how to define a stem cell; different definitions can be adopted that capture different features of interest. In the C. elegans germ line, stem cells can be defined by cell lineage or by cell commitment (‘commitment’ must itself be carefully defined). These definitions are associated with two other important questions about stem cells: their functions (which must be addressed following a systems approach, based on an evolutionary perspective) and their regulation. I review possible functions and their evolutionary groundings, including genome maintenance and powerful regulation of cell proliferation and differentiation, and possible regulatory mechanisms, including asymmetrical division and control of transit amplification by a developmental timer. I draw parallels between Drosophila and C. elegans germline stem cells; such parallels raise intriguing questions about Drosophila stem cells. I conclude by showing that the C. elegans germ line bears similarities with a number of other stem cell systems, which underscores its relevance to the understanding of stem cells.

Keywords: Caenorhabditis elegans, germ line, stem cells, commitment, transit-amplifying, mutations, systems biology

Introduction

The intense interest in stem cells transcends any particular definition of ‘stem cell’, and arises mainly from two expected functions: (a) the maintenance of genome integrity, and (b) the finely-controlled generation of a large number of a variety of specialized cell types from a small amount of homogeneous starting material.

How biological organisms perform these two functions is of great interest. From a scientific perspective, understanding how stem cells act as a tool for maintenance of genome integrity would provide insights into the evolutionary forces that derive from DNA damage. Furthermore, understanding the regulation of stem cell proliferation and differentiation would provide insights into the mechanisms of embryo development and adult organ homeostasis, and into the constraints that shape these mechanisms. From a therapeutic perspective, goals are to find means by which to minimize DNA damage, to minimize the incidence of cancer and birth defects, and perhaps to slow ageing. A further goal is to achieve rigorous control over the expansion and differentiation of cells that have been manipulated in vitro before in vivo delivery.

An approach based on molecular mechanisms has allowed an ever-expanding characterization of particular mechanisms by which genome integrity could be maintained, as well as of the wiring of stem cell gene regulatory networks. However, this knowledge forms a collection of parts that is by itself not sufficient to address the questions about stem cells formulated above. Indeed, stem cell function and regulation take place on the larger scales of cells, tissues and organisms; questions about evolutionary forces and constraints require consideration of populations and environments.

A systems-biology approach can bridge the gap between stem cell function and regulation at higher scales on the one hand, and the strategies and molecular mechanisms that are relied on for function fulfilment and regulation on the other hand. In brief, such an approach can start from expected stem cell functions and establish a list of candidate strategies and mechanisms these functions could rely on (for concrete examples, see section ‘Why have stem cells: prevention of mutation accumulation?’ in the Supporting information). These candidates can then be used to guide experimental exploration of stem cell systems. Confirmation of the presence of the candidate strategies, as well as a ‘reasonably-optimal design’ of the system, can be used to infer that the corresponding functions are of importance to the organism [1,2]; importance of the functions can be further assayed by experimental evolution studies. In summary, system functions can be used to guide exploration of the immense set of possible strategies and molecular mechanisms, while strategies and molecular mechanisms can in turn be used to infer more about system functions. An important future goal is to use the choice that is made between different possible strategies and mechanisms to outline the constraints and evolutionary forces to which organisms are subjected.

Despite the expectation that stem cells play a pivotal role in maintenance of genome integrity and finely-controlled generation of differentiated cells, it is not obvious that stem cell-based systems carry out these functions better than other systems would. This is partly because the detailed mechanisms by which stem cells could contribute to these functions are still unclear. Another issue is the definition of the term ‘stem cell’. A commonly-used definition of a ‘stem cell’ is that it proliferates, undergoes sustained self-renewal and contributes differentiating descendants; but this simple definition raises a number of conceptual problems [3,4]. There is no more detailed definition of ‘stem cell’ that is universally agreed upon [3,4], but two commonly expected characteristics of stem cells related to functions (a) and (b) listed above are that they should be at the top of a strongly hierarchical structure in terms of both cell lineage and differentiation commitment.

According to the cell lineage view, stem cells should contribute more descendants, over a much larger time span, than other proliferating cells. In the following, such stem cells will be referred to as ‘actual stem cells’; they should display self-maintenance and production of a large number of differentiated progeny (following the definition of [3]), and in addition should be at the top of a hierarchy of proliferating cells. This definition excludes progenitor cells that are part of a large pool of equivalent cells that lacks strong lineage hierarchy (as for example in the model proposed by [5]). According to the differentiation commitment view, stem cells should be uncommitted with respect to the time and outcome of differentiation, with stem cell descendants having more restricted potential. This criterion corresponds loosely to the definition of ‘potential stem cells’ [3], which have the potential to display actual stem cell behaviour, but both commitment and potential are difficult to define precisely (see below).

Closely associated with the notion of stem cells is that of transit-amplifying cells. Similarly to stem cells, transit-amplifying cells can be defined by lineage (ie cells that divide a limited number of times), or by differentiation commitment (ie cells that are committed to differentiation).

Here I first review the existence and control of stem cells in the C. elegans germ line according to lineage and differentiation commitment. These two views on stem cells lead to different views on transit-amplifying cells, and a new definition of transit amplification in Drosophila suggests parallels in regulatory mechanisms between C. elegans and Drosophila germ lines. I then ask whether the C. elegans germ line is at an evolutionary optimum, how stem cells as defined by lineage could contribute to the optimal achievement of function (a), whether the existence of such stem cells could be a secondary epiphenomenon, and how stem cells as defined by commitment could contribute to optimal achievement of function (b). I conclude by discussing the relevance of the C. elegans germ line as a model to investigate some of the evolutionary forces acting on biological organisms, and to understand the control of organs that share geometrical and regulatory designs with the C. elegans germ line.

Stem cell location and control

The C. elegans germ line provides a stem cell model that is exceptional in its ability to be observed in vivo, and by the extensive characterization of its gene regulatory network (for an introduction to the germ line, see [6]). C. elegans has two sexes: males and self-fertilizing hermaphrodites, which produce a fixed number of sperm during development before switching to oogenesis. The hermaphrodite gonad consists of two arms with identical structure (see Figure 1 for gonadal arm organization).

Figure 1
Distal end of a C. elegans gonadal arm. (a) Ray tracing rendering of germ cells (blue, Hoechst staining) and of the distal tip cell (green, lag-2::gfp), which acts as a niche for distal germ cells. Cells in the mitotic region, located distally, are all ...

The C. elegans germ line population grows exponentially during larval development, from two precursors set aside early during embryogenesis to about 1000 cells/arm upon the beginning of adulthood [7]. Germ cell number remains stable in adults for at least 5 days [8], a large proportion of the C. elegans lifetime. As larval development proceeds, a wave of meiotic entry spreads in a proximal-to-distal direction, so that in adults cell proliferation is limited to the distal end of each gonadal arm, within the ‘mitotic region’ where all cells are actively cycling (Figure 1) [8]. The mitotic region also maintains a constant number of cells (roughly 220) at least 5 days into adulthood, despite loss to cell differentiation [8], and thereby undergoes extensive turnover during the lifetime of a worm [9]. Most studies were performed 24 h after the last larval stage (L4), at which point the mitotic region spans about 20 cell rows on the distal–proximal axis (see Figure 1c–d for row numbering). Cells in the mitotic region are likely displaced in a distal-to-proximal direction as they differentiate, resulting in distal-most cells behaving as actual stem cells; stem cell behaviour as defined by commitment seems to extend over a much larger region (see below).

The mitotic region is specified and maintained by the distal tip cell (DTC; [10]), which caps the distal end of the germ line (Figure 1) and forms a cellular niche by providing a Notch signalling ligand ([11]; for a review of Notch signalling in C. elegans, see [12]). Downstream of the distal tip cell and Notch signalling, an intricate gene regulatory network computes the decision that is made by cells in the mitotic region between continued proliferation and differentiation by entry into meiosis [13,14]. Niche and regulatory network do not seem to specifically control actual stem cells, but could rather act by defining a zone of uncommitted cells and a zone of transit-amplifying cells (see below).

Stem cells as defined by lineage: actual stem cells

The spatiotemporal pattern of mitotic events in the C. elegans larval germ line varies from worm to worm, as assayed by real-time tracking of cell divisions in live larvae ( [15,10]; tracking of germ cell divisions in adults has not been reported). In other words, germline lineage is indeterminate (as defined by [16]). It is unfortunately too challenging to track all divisions in a given individual with current techniques, and lineage indeterminacy makes it impossible to collate partial data obtained in different individuals. Germline lineage is therefore unknown in any given individual.

The lack of lineage data makes it difficult to directly assay the existence and location of actual stem cells. The maintenance of a constant number of cells in the mitotic region despite loss to cell differentiation does not prove per se the existence of actual germline stem cells. Indeed, organs such as the pancreas, liver and kidney appear to be normally maintained by proliferation of differentiated cells without stem cells at the top of a highly structured lineage ( [17]; note, however, that injury can lead to the mobilization of facultative progenitors [18,19]); similarly, mouse tail epidermis has been proposed to be maintained by proliferation of a large pool of equivalent progenitors [5]. However, consideration of cell movement in the gonad does suggest the existence of actual stem cells. ‘Mitotic pressure’ created by cell proliferation cannot be released distally, as gonadal arms are closed at their distal end and maintain a grossly similar shape during adulthood. Cells are continually displaced in a proximal direction (at least once they have stopped proliferating [8]), likely because of the mitotic pressure created by their distal neighbours. Groups of cells labelled by BrdU incorporation travel down the gonad as a loose group (Figure 6 of [8]), suggesting limited cell mixing along the distal–proximal axis. Continuous displacement of cells in a proximal direction suggests that only cells in the distal-most row of the gonadal arm can contribute descendants maintained in the mitotic region over time. According to this simple idea, all other cells and their descendants should eventually be pushed out of the mitotic region by their distal neighbours, making it impossible for them to be actual stem cells (it is also possible that cells are pulled by the proximal germline instead of, or in addition to, being pushed by distal mitotic pressure, which does not change the argument). The first row (comprising about five cells [8]) would thus always maintain descendants within the mitotic region, unlike the other 19 rows in the mitotic region (comprising about 215 cells). The first row would thus be at the top of a strongly hierarchical lineage, making cells in the first row actual stem cells as a group [14].

Some complications arise, however, when interpreting the currently available experimental data to infer the presence of actual stem cells in the C. elegans gonad. It is not known to what extent the relative position of germ cells can be rearranged and whether, for example, cells normally slide past one another. Germ cells are only partly cellularized and form a syncytium by maintaining ‘intercellular bridges’ (or ring canals) to a common rachis ( [20, 21, 22]; this is a germ cell behaviour shared across species [23]). These bridges perhaps constrain relative movement somewhat. If the mitotic region were fluid and easily allowed for local cell re-arrangements, it would then be possible that:

  • Cells could at times find their way back to the first row from more proximal rows, displacing first row residents. Note that this does not contradict the population-level distal-to-proximal movement observed by [8], proximal to the mitotic region.
  • Cells could undergo extensive mixing within the mitotic region. Note again that this does not contradict the limited proximal–distal mixing observed by [8] in proximal cells that have entered meiosis, because cell divisions would provide opportunities for rearrangements. If mixing was sufficiently strong, there would effectively be no actual stem cells. However, such an extreme situation seems unlikely, especially since little lateral mixing occurs in the intestinal crypt, whose structure is comparable to that of the C. elegans germ line (see eg Figure 2 of [24] or Figure 4 of [25]).
  • Some cells could be actively migrating. Active cell migration has been proposed as an alternative to mitotic pressure, to explain cell movement within the intestinal crypt [26,27]. However, imaging of fixed C. elegans germ lines has not provided any evidence of cellular structures involved in cell migration.
  • Conversely, some proliferating cells other than cells at the first row could stay at a fixed position. The presence of a basal lamina [22] would provide an opportunity for anchoring.

It should also be noted that not all cells in the first row need be actual stem cells in all individuals. For example, consider two cells A and B in the first row; if in a given individual cell A divides and displaces cell B from the first row (Figure 1d), then cell B is not an actual stem cell. Such displacement events might occur randomly, making it impossible to refine the position of actual stem cells further than row 1.

In summary, cell-tracking studies will be required to confirm actual stem cell behaviour of the first row of the C. elegans gonadal arm and exclusivity of actual stem cell behaviour to the first row (in the following it will be assumed for simplicity that both these inferences are true). Cell-tracking studies will also be important to the understanding of the mechanism controlling differentiation (see section ‘Stem cells as defined by commitment’). Promising experimental techniques include photoactivable labelling of individual cells, and inducible recombination (eg as used by [5]). These techniques will not provide exhaustive germ cell lineage in individual worms, but should make it possible to delineate the behaviour of key groups of germ cells, such as the group of cells in row 1.

Control of actual stem cell lineage: symmetrical and asymmetrical divisions

A long-standing problem in stem cell biology is how actual stem cell progeny are made to diverge in their fate. After division of an actual stem cell, a daughter cell can leave the actual stem cell state and generate further progeny that will all differentiate within a short time frame (on the scale of the organism’s life span), or a daughter cell can maintain an actual stem cell identity. Divisions after which one daughter cell maintains a stem cell identity and the other daughter cell does not are defined retrospectively as asymmetrical divisions in terms of lineage. It is a requirement that some (but not necessarily all) of the stem cell divisions be asymmetric in terms of lineage, so that stem cells can contribute differentiated progeny while also having progeny that maintain a stem cell status.

It has been proposed that, in some stem cell systems, lineage asymmetry between two daughter cells is caused by an asymmetry that can be defined by the physical characteristics of the division of the mother cell. That division can be asymmetrical in terms of:

  1. Position of the daughter cells with respect to external regulators, such as a stem cell niche, a very short time after division: eg when one daughter cell is positioned so as to maintain extensive physical contact with the niche (and maintains stem cell status because of continued signalling from the niche) and the other daughter is not (and loses stem cell status because of a reduction or a loss of niche signalling). Asymmetry with respect to position in the niche is a common occurrence in the C. elegans germ line (Figure 1; [8]): divisions of first-row cells along a plane that is perpendicular to the gonad axis likely give rise to one daughter cell in the first row and one daughter cell in the second row. Importantly, however, this asymmetry is likely to make daughter cells diverge in fate because the cell in row 2 will end up being pushed away, and not because of differences in niche signalling between rows 1 and 2 (which are both embraced by the cellular niche).
  2. Partition of cytoplasm (so that daughter cells are created unequal in size) or of specific organelles or regulatory molecules during division. Such asymmetric divisions are not a requirement for stem cell systems (see also [28]). Differences in daughter cell size have not been reported in the C. elegans germ line (which could be due in part to the lack of live imaging of germ cell division), and neither has asymmetric partition of regulators (which again could be due to lack of live imaging, or to the relevant regulatory molecules and differentiation markers being still unknown).
  3. Expression of differentiation markers a very short time after division. Asymmetric expression in daughter cells could stem from asymmetric segregation from the mother (see point 2), or from differential synthesis or degradation in the daughter cells (due to differences in signals received by the daughter cells because of an asymmetry in their positions; see point 1). Again, such asymmetric divisions are not a requirement for stem cell systems and have not been observed in the C. elegans germ line.

The only physical asymmetry in the division of putative actual stem cells observed in the C. elegans gonad thus occurs when a cell in row 1 divides into a daughter cell that stays in row 1 and a daughter cell that goes to row 2 (gonads seem to be radially symmetrical, so that a division in row 1 that gives rise to two cells that stay in row 1 is a symmetrical division in terms of position). Such asymmetry occurs naturally and cell fate after such a division does not need to be controlled by an active regulatory mechanism: because of the tubular structure of the C. elegans germ line, only daughter cells that end up in the first row after division can be maintained as actual stem cells.

Symmetrical actual stem cell divisions are of great importance for clonal dynamics, because they allow a mutant clone to expand and push others out of the niche. These divisions could allow the spread of nefarious mutations involved in carcinogenesis [29] or birth defects. Stem cell replacement by neighbouring actual or potential stem cells has been inferred to occur in the Drosophila ovary [30,31], in the mammalian testis [32] and in intestinal crypts [33,34]. In the C. elegans germ line, the plane of cell division in row 1 is frequently parallel to the axis of the arm [8], in which case both daughter cells are expected to stay in the first row, making for a symmetrical division. When such a division occurs, another cell is presumably pushed out of the first row — an idea that will have to be confirmed by cell-tracking studies.

In summary, actual germline stem cells seem to undergo divisions that can be symmetrical or asymmetrical in terms of lineage. It is not known whether and how the proportion of divisions that are symmetrical or asymmetrical is actively controlled. Interestingly, some mutations can increase the likelihood of stem cells being retained in the Drosophila ovary niche and displacing other stem cells [35]; it is not known whether such active competition also occurs in the C. elegans germ line.

Stem cells as defined by commitment

‘Commitment’ is a thorny issue (see eg [16]). The ability of nuclei and whole cells to be reprogrammed by various means [3638] makes it a safe bet that no cell is ever committed to a specific identity, in the sense that there will always be some experimental treatment that can change its identity ([39]; see also [40]). The feasibility of reprogramming makes it necessary to tailor definitions of commitment to particular aims. For therapeutic purposes, any practical experimental treatment is permissible. To understand the in vivo regulation of stem cell differentiation, commitment tests are best based not on exposure to artifical stimuli or manipulation of gene regulatory networks, but rather on exposure to environments that cells could naturally be faced with (eg by experimentally altering cell position within an embryo [4143]), or exposure to a ‘neutral’ environment (eg by extracting cells from an embryo and culturing them in vitro [44,45]).

What, then, are ways to test germ cell commitment? Possible assays include:

  1. Grafting cells from the proximal mitotic region back to the first row, and assaying whether they behave as stem cells by maintaining the mitotic region. An experimental limitation of the C. elegans germ line model system is the current lack of methods to transplant germ cells (or culture them in vitro). This lack is shared by many model systems, with the notable exceptions of haematopoiesis — which by nature contains circulatory cells that are naturally more amenable to isolation and engraftment — and mouse spermatogenesis.
  2. Temporarily removing niche signalling, forcing the first to row to proceed along differentiation. Restoring niche signalling would then lead to a situation where cells that have embarked on differentiation are in row 1, similar to a situation in which proximal cells have been grafted into row 1 (as proposed above). Results from such an experiment could then be used to infer the differentiation stage at which cells become ‘committed’ to differentiation. In the Drosophila germ line, such experiments have shown that differentiating stem cell descendants can revert to a stem cell state ( [46,47]; see section ‘Stem cells: Drosophila versus C. elegans’ for further details). Although genetic tools are available in C. elegans to temporarily remove niche signalling, differentiation in the mitotic region does not seem to proceed in discrete, easy-to-identify steps. It might be possible with such experiments to infer that differentiating C. elegans germ cells can revert to a stem cell state, but it would be difficult to pinpoint the precise stage at which that competence is lost. In addition, it could be argued that extensive backward movement within the germ line is unlikely to happen naturally, and that this experiment is therefore not a test of commitment under natural conditions, as defined above.
  3. Blocking cell movement in the mitotic region, and asking which cells proceed with differentiation. Committed cells would differentiate without needing further positional cues. Definition of commitment by this assay is less restrictive than by assays 1 and 2: cells that differentiate in the absence of movement (and are therefore committed according to assay 3) might revert to a stem cell-like state if moved back to the first row (and therefore not be committed according to assays 1 and 2), whereas a cell that is unable to revert to a stem cell like state when moved back (and is therefore committed according to assays 1 and 2) would likely differentiate if left at the same position (and would therefore be committed according to assay 3). An interpretation of this form of commitment in terms of tipping the balance of the regulatory network between mitosis and meiosis is proposed below.

It should be noted that all these assays assume for simplicity that commitment is a cell-autonomous character. An intriguing complication is that germ cells form syncytia in many species. The role of these syncytia is an important and unresolved question.

It has been proposed that, in principle, the C. elegans mitotic region could be divided between stem cells, which would not be committed to differentiation, and transit-amplifying cells, which would be committed [9, 13, 4850]. However, no experiments have directly addressed that question. Among the assays proposed above, only the prevention of normal germ cell displacement from the distal end to the proximal end is practical at present; this can be achieved by blocking the cell cycle. This in effect blocks the change in extracellular context to which cells would normally be subjected by their displacement towards the proximal end of the gonad.

Upon blocking the cell cycle using a temperature-sensitive cell cycle machinery mutant or a chemical inhibitor, germ cells remain in an undifferentiated state in a distal zone of the mitotic region that encompasses about nine cell rows and 70 cells in total (Figure 1e–f; [51]). By contrast, cells of the mitotic region proximal to this zone do differentiate upon blocking the cell cycle, as assayed by differentiation markers such as GLD-1 and HIM-3 (Figure 1e–f), showing commitment to proceed with differentiation independently from further positional cues. Maintenance of the distal region in an undifferentiated state is dependent on continued Notch signalling from the niche: laser ablation of the distal tip cell or disruption of Notch signalling with a temperature-sensitive mutant lead to differentiation of distal cells. This suggests that cells in the nine most distal rows of the C. elegans gonad are potential stem cells, as they are not committed to differentiation until they have been displaced further proximally in the gonadal arm. This zone of potential stem cells was dubbed the ‘stem cell zone’ (SCZ; [51]).

After potential stem cells move past the ninth row and commit to differentiation, they still proliferate transiently, suggesting they are analogous to transit-amplifying cells in other model systems. For this reason, the zone proximal to the SCZ was dubbed the ‘transit-amplifying compartment’ (TAC; [51]). Note that it is possible that increased Notch signalling or displacement closer to the distal niche would revert these transit-amplifying cells to a potential stem cell state; however, this is a situation that is unlikely to happen extensively under natural conditions, because of the likely distal-to-proximal movement of the germ cells.

Control of uncommitted cells and their descendants: position and timing

The SCZ is likely specified by position with respect to the distal tip cell. This is because the DTC provides positional information to the gonad [10] and maintains the SCZ in an undifferentiated state [51]. How this positional information is provided and how it is read by the germline regulatory network are important questions for the future. One can envision that a decreasing extent of contact between DTC and germ cells ( [8]; Figures 1a, b), as well as intercellular diffusion of regulatory molecules within the syncytium, could create a gradient of Notch signalling or of downstream Notch effectors along the distal–proximal axis. The germline regulatory network would respond in an all-or-none fashion to a drop in signal intensity past a specific threshold, and change germ cell status from uncommitted cell to transit-amplifying cell.

According to a simple model, differentiation of TAC cells could be controlled by the time it takes the regulatory network to complete its switch from a stem cell-like state to a differentiated state [51]. Germ cells would keep proliferating from the time they leave the SCZ and enter the TAC to the time the switch is complete, at which point they would enter meiosis. The regulatory network would thus implement a differentiation timer.

Timers have been proposed to operate in a wide range of developmental contexts, including protozoan differentiation [52], oligodendrocyte differentiation [53], retinal neuron fate choice [54], the Xenopus midblastula transition [55], formation of the C. elegans vulva [56], Drosophila germline cyst formation [23] and vertebrate segmentation [57]. Many questions arise when considering these multiple timers:

  • Do timers rely on the number of cell divisions? When it has been possible to address that question, a picture has emerged according to which most timers can operate independently of the cell cycle ( [23, 52, 55, 56, 5861]; however, see [62]); this is consistent with the ability of a wide array of cells to differentiate at a point of the cell cycle other than G0 [6366]. Similarly, the C. elegans germline differentiation timer appears independent of cell division count [51].
  • What are the molecular mechanisms of timers? Even in the case of vertebrate segmentation, which was surmised long ago to rely on a timer (underlying the wavefront in the ‘clock and wavefront’ model [67]) that has since been the object of intense attention, many fundamental questions remain unanswered [68]. A strength of the C. elegans germ line as a model system is the extensive characterization of its regulatory network at both the genetic and biochemical levels. Because the germline differentiation timer is likely implemented by that regulatory network, this system could be one of the first where a differentiation timer is thoroughly dissected.
  • Are molecular mechanisms conserved between timers? This appears possible, as cyclin E has been associated with many of the timers mentioned above [53, 55, 56, 69]. A common feature of timers is that they could rely in part on post-transcriptional mechanisms. This is likely the case for timers in neurogenesis [70], in retinal cell-type determination [62], in C. elegans larval development [71] and also for the C. elegans germ line, because of the strongly post-transcriptional nature of its regulatory network [14,72,73].
  • Do timers run freely once triggered? The oligodendrocyte differentiation timer is modulated by extracellular signalling both in vivo and in vitro [74], which could also be the case for the retina timer [75], and it has been proposed that a component of the haematopoietic stem cell niche acts not only to maintain stem cells but also their differentiating descendants [76]. Similarly, the C. elegans germline differentiation timer could be externally modulated by regulator diffusion within the germline syncytium (such diffusion was proposed in the mammalian germ line by [77]). Regulator diffusion could allow influence of the distal tip cell to extend to all of the mitotic region. Diffusion could also allow local synchronization of timers, so that cells that entered the TAC and thus triggered their timer at the same time, but were separated along the distal–proximal axis because of cell proliferation, would resynchronize with their neighbours and avoid ectopic differentiation (it is not certain at present that such synchronization does occur: the extent of cell mixing in the TAC is unknown, and cells likely do not all enter meiosis at the exact same distance from the distal end but rather over a region spanning a few cell diameters [8], suggesting that synchronization could be of limited strength). It thus appears that modulation by the extracellular environment could be a common feature of differentiation timers.

In summary, regulation of differentiation in the C. elegans germ line relies on a subdivision of the mitotic region in two zones. The distal zone (SCZ) feeds cells into the proximal zone (TAC). The distal zone is maintained by the stem cell niche, while the proximal zone is controlled by a differentiation timer that appears to be modulated by the niche. The differentiation timer could share important features with timers identified in other systems. Extensive knowledge of the germline regulatory network will enable a dissection of the molecular mechanism of the timer, which should provide insights into other systems.

Stem cells: Drosophila versus C. elegans

Stem cells and transit-amplifying cells in Drosophila versus C. elegans

The structure of Drosophila testes and ovarioles is comparable to that of the C. elegans germ line [78]. Somatic cells located at the tip provide a niche required for germ cell proliferation [78,79]; cells appear to be displaced further and further from the niche as they progress towards gamete formation. Germ cells in physical contact with the niche have been identified as stem cells on the basis of lineage studies [80,81]; these stem cells display self-maintenance and production of a large number of differentiated progeny, providing a parallel with actual stem cells in C. elegans.

Having established a parallel between C. elegans ‘actual stem cells’ (as defined in this manuscript) and Drosophila stem cells (as defined in the Drosophila literature), one can next compare differentiation of stem cell descendants. In the Drosophila melanogaster male germ line, stem cell descendants that are displaced from the niche go through gonialblast and spermatogonium stages, during which they undergo four rounds of mitosis before entering meiosis and producing sperm (reviewed by [82]). In the Drosophila melanogaster female germ line, stem cell descendants go through cystoblast and cystocyte stages and also undergo four rounds of mitosis (reviewed by [83]). The four rounds of mitosis produce a 16-cell egg chamber; one of these cells becomes an oocyte, while the remaining 15 act as nurse cells.

Commitment of spermatogonia and cystocytes has been assayed by induction of stem cell differentiation with a transient block of niche signalling ( [46,47]; assay 2 in section ‘Stem cells as defined by commitment’). It was shown that cysts of up to eight cells, which had thus undergone all but one of the four mitoses that occur before gamete formation, could break up and contribute cells that reverted to a stem cell state and replenished the niche. Importantly, even though the precise origin of these reverting cells is unknown, it appears likely that the cysts contributing reverting cells were those juxtaposed to the niche, an abnormal position for this stage of differentiation. Other definitions of commitment, based on assays that do not expose differentiating stem cell descendants to much stronger niche signalling than they would normally experience, might show commitment at earlier points of differentiation and reveal important regulatory stages (see below).

It is thus not possible at present to directly compare differentiation commitment between Drosophila and C. elegans germ lines, because commitment has not been assayed in similar ways. Importantly, mitosis of gonialblasts and spermatogonia has been referred to as ‘amplificatory mitotic divisions’ [84] and ‘transit amplification’ [46, 79, 85]. Use of the term ‘transit amplification’ has been deemed inappropriate in the case of the Drosophila female germ line, because each cystoblast gives rise to only one egg chamber and divisions do not amplify gamete number [86]. It is crucial to note that transit amplification can be given two sorts of definitions, which parallel stem cell definitions based on lineage and commitment. For example:

  1. A definition based on lineage, requiring a short residence time of transit-amplifying cells and their descendants in a proliferative compartment (eg [87] defines transit-amplifying cells as ‘being produced by a precursor cell population and having a clearly limited, relatively short period of existence in the body’).
  2. A definition based on commitment, requiring cells to be committed to differentiating within a given time interval (eg [88] defines transit-amplifying cells as being ‘committed to produce mature cell lineages’). As noted above, commitment needs to be precisely defined; this might be difficult in the case of haematopoiesis, where the concept of transit amplification originated, because stem cells appear to be fluctuating entities [89,90]. A complication is also that commitment can refer to cell type instead of time of differentiation (eg erythroid-committed progenitor); there is some overlap between the two definitions because both commitments appear to take place within a short time interval in some systems.

These two definitions can designate very different sets of cells as transit-amplifying:

  • By definition (1), all rows of the C. elegans mitotic region except for the first row would be comprised of transit-amplifying cells. This is a consequence of the geometry of the gonad, which results in these cells eventually being displaced out of the mitotic region by descendants from the first row (see above). Definition (1) is the one that would correspond to the way ‘transit amplification’ has been used in the Drosophila literature.
  • By definition (2), only the proximal half of the C. elegans mitotic region is comprised of transit-amplifying cells, because that is the region where cells are committed to differentiation (commitment being defined by assay 3 in the section ‘Stem cells as defined by commitment’). Definition (2) is used in this manuscript in reference to C. elegans, because regulation of germ cells by the germline niche is deemed to be of more interest than the geometry of the gonad.

Importantly, transit-amplifying cells are sometimes associated with a sustained and fast cell cycle [87], while stem cells are expected to cycle more sporadically (with pauses in G0) or more slowly. Long-term retention of a pulsed DNA base analogue has, however, been shown not to be a universal property of stem cells [91,92], arguing against sporadic proliferation as a useful marker of all stem cells (label-retaining stem cells might be highlighted by studies involving reconstitution after injury [93]). Moreover, although benefits from slow stem cell proliferation can be envisioned (see Supporting information), and although fetal haematopoietic and C. elegans germline stem cells could cycle two-fold slower than more differentiated descendants ( [50, 94]; however, see [8] and Supporting information), one cannot assume that slow cycling is a general property of stem cells: for example, embryonic stem cells cycle very fast (noted by [95]). Finally, transit-amplifying cells need not cycle faster than stem cells: for example, Drosophila testis transit-amplifying cells cycle at the same rate as stem cells [96]. Differences in cell cycle therefore do not necessarily provide per se an indication of stem cell or transit-amplifying status.

In summary, lineage behaviour of C. elegans ‘actual stem cells’ and Drosophila ‘stem cells’ (as used in the Drosophila literature) is likely similar. Behaviours in terms of differentiation commitment cannot be directly compared for lack of experimental data, and an interesting question for the future is whether the Drosophila germ line has a ‘stem cell zone’ as defined for C. elegans. The next section explains how defining such a stem cell zone might provide insights on stem cell regulation.

Tipping the balance between stem cell state and differentiated state

It appears difficult to define Drosophila germline stem cells by a unique combination of gene product expression. Indeed, it would seem that gene products that have been shown by immunostaining to be expressed in stem cells are also expressed in gonialblasts or cystoblasts, and vice versa (Table 1 in [97]), with only a handful of potential exceptions.1 This holds both for genes associated with stem cell maintenance (eg pumilio and Dpp signalling targets) and for genes associated with differentiation (eg bag of marbles (bam), a gene promoting differentiation in both male and female germ lines [100103]; note, however, that bam does not localize to the cytoplasm in stem cells, unlike in gonialblasts and cystoblasts [101]). However, there are differences in levels of expression: expression of Pumilio and phosphorylation of Mad (a Dpp signalling target) decrease from stem cells to cystoblasts [86, 104106], while expression of Bam conversely increases from stem cells to cystocytes [101].

In the C. elegans germ line, the same principle holds of absence of stem cell-specific gene expression and graded expression of the same genes from stem cells to differentiated cells. The Pumilio homologues FBF-1 and FBF-2 are expressed throughout the mitotic region [48,107], as is GLD-1, a gene promoting differentiation (with a dramatic increase in intensity on the distal–proximal axis [108]) [109111].

A common feature in regulatory networks driving cell differentiation is cross-repression of genes promoting different cell fates; this cross-repression can give rise to switch-like behaviour (eg [112117]). The genes mentioned above only give a partial picture of the regulatory networks driving the decision between mitosis to meiosis, but a common pattern emerges from the interactions between these genes (Figure 2). In Drosophila, Dpp signalling inhibits differentiation in part by repressing bam expression [85,98,118], while Bam represses Dpp signalling [119]. In C. elegans, Notch signalling inhibits differentiation in part by repressing gld-1 [48,107], while GLD-1 represses expression of the Notch receptor [120]. In both instances, these cross-repressive interactions create a positive feedback loop that could force an all-or-none decision between stem cell state and differentiated state. Relative expression and activities of factors promoting or antagonizing differentiation are likely to determine the point at which the balance between stem cell state and differentiation is tipped (for the formulation of the idea of a balance, see eg [121] for Drosophila and [122,123] for C. elegans).

Figure 2
Dramatically simplified outline of the regulatory networks controlling stem cell differentiation in the C. elegans hermaphrodite germ line (a) and in the Drosophila female germ line (b). Cross-repression between niche signalling promoting stem-cell maintenance ...

Once the balance is tipped, because of weakened niche signalling as cells are displaced by proliferation, it would take some time for the regulatory network to complete the switch from a mitotic to a meiotic state; mitosis could still occur during this transition, which would correspond to a transit-amplifying state. In order to understand how the decision between mitosis and meiosis is computed, it is desirable to know where in the germ line the balance is tipped. In terms of the commitment language used above (assay 3), cells where the balance has not been tipped are not committed to differentiation and correspond to the ‘stem cell zone’ defined experimentally in C. elegans. Similar experiments could define a stem cell zone in Drosophila.

Definition of a ‘stem cell zone’ in Drosophila might reveal regulatory stages that have been hard to address. For example, it has been proposed that differentiation of stem cell daughters to cystoblasts proceeds through an intermediate ‘pre-cystoblast’ stage, characterized by its dependence on Bam activity to proceed with differentiation [102] and by its requirement for the gap-junction protein Zpg ( [86]; see also [119]). This intermediate is not known to express a different set of proteins from stem cells and cystoblasts [86], and its detailed characterization has been elusive. An intriguing possibility would be for commitment in the Drosophila germ line to happen at the pre-cystoblast stage, if commitment is defined by blocking movement in the germ line.

Finally, the number of mitotic divisions that Drosophila germline stem cell descendants go through after leaving the niche varies between species [124,125] and can be altered by changes in dosage of cyclin E [69] and encore ( [23,126]; Encore facilitates cyclin E proteolysis [127]). An attractive possibility to explain these variations is that exit from the mitotic cycle is controlled by a differentiation timer similar to the one proposed in C. elegans, and that mutations alter the rate of progression of the timer. Interestingly, somatic cells other than the niche influence differentiation in Drosophila males [128], suggesting that such a differentiation timer could be modulated by external factors, just as in C. elegans (note, however, that it is not clear that the over-proliferating cells described by [128] would correspond to a stage where the differentiation timer has not been started).

In summary, geometrical and regulatory parallels between the C. elegans and Drosophila germ lines suggest that control of differentiation by time and position proposed in C. elegans could be applicable to Drosophila.

The second part of this review addresses stem cell function, and compares the C. elegans gonad to other organs in which stem cells have been characterized. Because of space constraints, the second part can be found online as Supporting information. A full manuscript containing both parts can be downloaded from http://cinquin.org.uk

Supplementary Material

cinquin supporting

supporting

supporting 3

Acknowledgments

I am grateful to Judith Kimble for her support and to members of her laboratory in general, and Sarah Crittenden in particular, for many discussions and free sharing of data and ideas. Judith Kimble, Sarah Crittenden and Amanda Cinquin contributed comments that greatly improved this manuscript; any remaining shortcomings are my responsibility.

Footnotes

No conflicts of interest were declared.

1Phosphorylated Mad is present in male stem cells but not in gonialblasts (Figure 3D in [98]); because Dad–lacZ expression suggests that Dpp signalling could be active in gonialblasts, one explanation suggested by the authors is that phosphorylated Mad might be present below the detection threshold. DE-cadherin is expressed in stem cells but seems to be absent from their immediate descendants [99].

Supporting information

Supporting information may be found in the online version of this article.

Teaching Materials

Power Point slides of the figures from this Review may be found in supporting information.

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