In the foregoing sections, we described mechanisms that specify bona fide C. elegans
stem cells, the germline stem cells, and considered regulation of self-renewing stem cell-like lineages that arise during post-embryonic development of the seam cells. A key problem in stem cell biology is to understand how stem cells maintain multipotentiality and avoid committing to a unique differentiated fate. With the knowledge of the molecular regulatory circuitry that allows stem cells to remain pluripotent, and how this circuitry becomes modified when cells switch from a multipotential state to a committed pathway of differentiation, it may be possible to produce stem cells from virtually any differentiated cell type. A major technological advance in this field was achieved with the discovery of methods for generating induced pluripotent (iPS) cells from fully differentiated cells of adult animals (Takahashi and Yamanaka, 2006
; Huangfu et al., 2008
; Nakagawa et al., 2008
) by expressing just a single factor, Oct4 (Kim et al., 2009a
; Kim et al., 2009b
), in neural stem cells. While production of iPS cells is a powerful method that promises to lead to the creation of a great variety of new stem cell types for clinical applications, there is much to be learned about the molecular processes that distinguish multipotential stem cells and their committed, differentiated descendants and how such processes may be reversed or altered, resulting in cellular transdifferentiation. This information may make it possible to reprogram fully differentiated cells into new cell types which can then populate functioning tissues. Using C. elegans,
it has been possible to analyze the steps that occur during natural transdifferentiation, regulatory events that repress somatic differentiation and maintain pluripotency during germline development, and molecular processes that convert pluripotent progenitors to cells of restricted differentiation potential during embryogenesis.
Natural transdifferentiation during C. elegans development: an epithelial-to-neural transformation
While the phenomenon of transdifferentiation has been known for many years (Pritchard et al., 1978
; Okada, 1980
; Slack and Tosh, 2001
; Slack, 2007
), very few examples of bona fide
transdifferentiation have been observed during normal animal development. For example, the process of larval metamorphosis, in which entire new tissues or organs are born in a differentiated animal, does not necessitate that fully differentiated cells of one type become transformed into cells of an altogether different cell type; rather, the newly differentiated tissues often arises from uncommitted progenitor or stem cells that had been set aside at earlier developmental stages (Cohen, 1993
). Genuine transdifferentiation is most convincingly demonstrated only when a cell is observed continuously and found to convert from one fully differentiated cell type to another. Such observations require cell lineage analysis, a technique that has been accomplished par excellence
with C. elegans
, in which the history and fate of every somatic cell observed continuously through development have been documented. The conversion of the “Y” rectal epithelial cell into a neuron, called PDA, during C. elegans
larval development (Jarriault et al., 2008
), is among the clearest examples of a true transdifferentiation event in any animal.
The Y cell, born during embryogenesis, is an essential structural cell of the rectal epithelium in the newly hatched larva (Sulston and Horvitz, 1977
; Sulston et al., 1980
; White, 1988
), and displays all the characteristic morphological features of a fully differentiated epithelial cell (Jarriault et al., 2008
). As post-embryonic development proceeds through the L2 stage, the Y cell withdraws from the rectum, migrates away from the rectal region, and apparently transdifferentiates into the PDA motor neuron, which projects processes that synapse with other neurons by the L3 larval stage. Significantly, this transdifferentiation process occurs in the absence of division of the Y cell; rather, it results from the complete remodeling of an extant post-mitotic epithelial cell into a neuron. Another lineally unrelated cell, P12.pa, which is born shortly before this event is initiated, replaces the departing Y cell in the rectal epithelium. Concomitant with its morphological transdifferentiation, this cell loses expression of all tested epithelial-specific markers, including proteins involved in epithelial polarity and transcription factors that specify epithelial fates, by the time it has become a neuron. Moreover, the trans-fated PDA neuron expresses a number of neuron-specific genes that are not detectable in the Y cell before this event has occurred. The transformation of a Y epithelial cell into a PDA motor neuron has been divided into five phases: establishment of Y cell identity, establishment of competence to undergo transdifferentiation, retraction from the rectum, migration of Y from the rectum, and establishment of PDA identity. Dissecting the molecular and cellular events that direct the transformation of Y into PDA will help to unveil the mechanisms underlying natural transdifferentiation and cellular plasticity.
Several experiments in which local cellular interactions were interrupted, including ablation of cells surrounding the Y cell, failed to prevent the Y-to-PDA transdifferentiation event; moreover, blocking the normal anteriorward migration of the erstwhile Y cell does not abrogate its transdifferentiation into a PDA neuron. Thus, region-specific cues do not appear to be essential for this event to proceed, which may be directed exclusively by cell-intrinsic programs. Indeed, two transcription factors, the homebox protein EGL-5/Abd-B, and the zinc finger transcription factor SEM-4/spalt are required for transdifferentiation of Y into PDA: loss of either in a Y cell causes it to remain epithelial throughout development and prevents formation of the PDA neuron. However, while essential, these factors are not sufficient for transdifferentiation and are therefore not specific to the process, as they are also expressed in other epithelial cells that do not transdifferentiate. Thus, EGL-5 and SEM-4 appear to function early by making Y competent for transformation into PDA. Though no evidence for cell-cell signaling in the transdifferentiation event has been obtained, the identity of the Y cell is specified in part by a LIN-12/Notch-dependent lateral interaction between the progenitors of Y and a neuron called DA9. Removal of lin-12
function causes what would normally have become the Y cell instead to adopt the fate of the DA9 neuron (Greenwald et al., 1983
). This signaling through LIN-12/Notch also appears to impart to the Y cell the ability to transdifferentiate during later development, as constitutively active LIN-12 results in production of an extra Y cell, from what would normally be the DA9 neuron. Along with the normal Y cell, this ectopic Y transdifferentiates into PDA later in development. It is not yet known whether LIN-12 endows the Y cell with the general susceptibility for transdifferentiation or instead specifically allows it to become only PDA as development ensues. However, it is not the case that all conditions in which an extra Y-like cell is generated allow Y to transdifferentiate into PDA: in egl-38(−)
mutants, in which the rectal cells U and B, respectively, are converted to Y-like cells based on morphology and marker expression, extra PDAs are not generated. Thus, other conditions must be met to predispose the Y cell to undergo later transdifferentiation that are distinct from those that dictate at least some Y-specific characteristics. Finally, it is unclear whether Y dedifferentiates before it becomes PDA or instead undergoes direct transdifferentiation without a dedifferentiation intermediate. If the former is correct, Y may be subject to reprogramming into other cell types when presented with another lineage-specific fate directing factor, a possibility that has not yet been tested.
Control of totipotency and exclusion of somatic development in the germline
Germ cells are capable of giving rise to an entire organism and hence may be viewed as the ultimate totipotent stem cells. While the mechanisms that specify the embryonic germline progenitors, the primordial germ cells (PGCs), vary between species, they share several common characteristics. The most striking of these commonalities is the transcriptional quiescence of early germ cells mediated by direct repression of RNA Pol II, as seen in C. elegans
and Drosophila, or by silencing mediated in part by the transcriptional repressor Blimp1(Nakamura and Seydoux, 2008
). These regulatory mechanisms result in total repression of all somatic programs of differentiation and also contribute to the maintenance of totipotency of the PGCs.
In addition to regulation at the transcriptional level, post-transcriptional regulatory processes, mediated in part by miRNAs, are of crucial importance to the development of germline stem cells (Gangaraju and Lin, 2009
), as has also shown to be the case with embryonic stem cells (Gangaraju and Lin, 2009
; Qi et al., 2009
). Repression of let-7
miRNA function by the LIN-28 RNA binding protein, as first observed in the stem cell-like divisions of the C. elegans
seam cells, was also found to be critical for germ cell specification and maintenance (West et al., 2009
); moreover, LIN-28 overexpression is associated with increased proliferation, leading to germ cell tumors (Viswanathan et al., 2009
; West et al., 2009
). This system is also relevant to reactivation of pluripotency in differentiated cells: LIN-28, when coexpressed with the transcription factors OCT4, SOX2, and NANOG is sufficient to reprogram human fibroblasts into ES–like induced pluripotent stem cells (iPS). Likewise, totipotent PGCs continue to express the pluripotency-determining genes Nanog
(Seydoux and Braun, 2006
). This close relationship between germline and embryonic stem cells is underscored by expression profile studies of mRNAs and miRNAs (Zovoilis et al., 2008
), which suggest that ES cells are most closely related to GSCs (Zwaka and Thomson, 2005
). Further, ES cells, germ cells and PGCs all can give rise to teratomas, rare germline tumors containing differentiated somatic cells representative of all three germ layers, suggesting a conserved pluripotency module.
The discovery of teratomas in the C. elegans germline offers new perspectives into the molecular circuitry that regulates totipotency. Ciosk et al. discovered that GLD-1 and MEX-3, two RNA-binding translational repressors involved in regulating GSC proliferation, also function together critically in maintaining germ cell totipotency by repressing somatic differentiation programs. Simultaneous removal of both factors results in the appearance of germline teratomas, containing differentiated cells characteristic of all three germ layers. Thus, translational repression of presumably many target transcripts by MEX-3 and GLD-1 is a key mechanism that prevents somatic differentiation, and maintains pluripotency, in the developing germline.
MEX-3 and GLD-1 function in part by repressing translation of somatically active transcription factors, such as the mesectoderm-promoting caudal-type homedomain protein PAL-1 (Ciosk et al., 2006
). In addition, elimination of GLD-1 function results in inappropriate translation of its direct target, the message encoding CYC-E (cyclin E), resulting in mitotic reentry of normally meiotic cells and premature activation of somatic gene expression (Biedermann et al., 2009
). Activation of somatic gene expression is not a consequence of inappropriate cell proliferation per se
, but is a consequence of activation of the CYC-E/CDK2 complex, as somatic gene transcripts continue to be expressed even when germ cell proliferation is blocked. A similar requirement for a cyclin/cdk complex, specifically cyclinA2/CDK2, has been reported for transcriptional activation of embryonic gene expression in the one-cell mouse embryo (Hara et al., 2005
How might inappropriate activation of cyclin/CDK complexes result in somatic gene expression in the germline? While cyclins and cdks were initially identified as cell cycle regulators, the known repertoire of their action has greatly expanded. Cyclin/cdk complexes have been shown to influence transcription by directly regulating specific transcription factors and generally by phosphorylating Ser2 and Ser5 in the carboxy-terminal domain (CTD) of RNA Pol II. These complexes have also been implicated in regulating splicing through the phosphorylation of splicing machinery components. These observations indicate that coordination of the regulatory machinery for the cell cycle, translation, and transcription is critical for regulating germ cell totipotency and for repression of somatic differentiation in the germline.
The multipotency → commitment transition in the early C. elegans embryo
Given the close relationship between pluripotent stem cells and early embryonic cells, much can be learned about the mechanisms controlling stem cell pluripotency by studying the plasticity of cells in early embryos. In the early C. elegans
embryo, progenitor cells with distinct lineage identities are born at each round of cell division, beginning at the first cleavage (Laufer et al., 1980
; Sulston et al., 1983
); there are no fields of equivalent self-renewing cells that are subsequently induced to adopt more specialized fates. Moreover, the stereotypic pattern of cell divisions and fates reveal a deterministic program of development (Sulston et al., 1983
). Combined with the lack of a system for cell culture, C. elegans
embryonic development might therefore seem to be poorly suited for the study of stem cell pluripotency and self-renewal. However, a number of studies have demonstrated that, while specification of distinct differentiation pathways apparently occurs very early in embryogenesis, cells nonetheless maintain pluripotency throughout much of the first half of embryogenesis as evidenced by their ability to be reprogrammed into alternative pathways of development when forced to express cell fate regulators that normally function in different lineages. Later in embryonic development, cells become restricted in their capacity to become redirected down alternative developmental pathways and firmly commit to their appropriate differentiation programs. This transition from multipotency to a state of restricted differentiation in the C. elegans
embryo provides a useful system for probing mechanisms that control pluripotency.
The evidence for a dramatic switch from a developmentally plastic to a committed state during embryogenesis has been obtained in a variety of cell fate reprogramming experiments. The five somatic and one germline “founder cells,” each which transmits a distinct cell cycle clock to its descendants and gives rise to a unique set of cell types and lineages, are born during the first several embryonic cell divisions. Ectopic expression of the END-1 GATA-type transcription factor, which is normally expressed only in the E founder cell lineage shortly after it is established (Zhu et al., 1997
), causes virtually all cells in the early embryo to become reprogrammed and to differentiate into intestine (endoderm) (Zhu et al., 1998
). In extreme examples, virtually every cell in terminal embryos differentiates into an intestinal cell, at the expense of all mesodermal and ectodermal differentiation. Thus, although only the E lineage normally engenders endoderm, every somatic cell in the early embryo has the capacity to do so. Similarly, ectopic expression of the PHA-4/FoxA transcription factor, which is essential to establish organ-specific identify of the various cells of the pharynx (Horner et al., 1998a
; Kalb et al., 1998
), leads to ectopic production of pharyngeal tissues and repression of non-pharyngeal differentiation; however, in contrast to the experiments with END-1, only a subset of embryonic cells are responsive to PHA-4-directed reprogramming (Horner et al., 1998b
). Subsequent experiments have revealed that early embryonic cells can also be reprogrammed into epidermal and other epithelial cells (Gilleard and McGhee, 2001
; Quintin et al., 2001
), as well as body wall muscle cells (Fukushige and Krause, 2005
), when challenged with the appropriate cell-fate-promoting transcription factors. The competence of cells of the early C. elegans
embryo to be redirected from their normal developmental fates into cell types representing all three germ layers demonstrates that they are genuinely pluripotent.
Studies in which the ectopic expression of specification factors is temporally varied demonstrated that embryonic cells are competent to be reprogrammed only during a restricted window of time, beyond which they become refractory to reprogramming (Horner et al., 1998a
; Zhu et al., 1998
; Gilleard and McGhee, 2001
; Quintin et al., 2001
; Fukushige and Krause, 2005
). This window of competency lasts until approximately 3 hours after the first cell division, during which dramatic changes in gene expression are occurring as a result of the widespread mobilization of differentiation programs throughout the embryo (Baugh et al., 2003
; Yuzyuk et al., 2009
). This period of developmental plasticity ends shortly after the founder cell identities are established and lineages become restricted to undergo differentiation into particular cell types (e.g., nervous system, epidermis, muscle, pharynx, and intestine). It is not known whether this developmental plasticity ends at precisely the same time in each lineage or, as seems likely based on the differences in their times of birth and lineage specification, the boundaries of the competency window vary somewhat between founder cell lineages. Further, it is not known whether the ability to become reprogrammed to a variety of cell types is equally distributed among the founder cell lineages. Nevertheless, the observation that the window of susceptibility to reprogramming is similar regardless of the cell fate specification factor used points to existence of a major transition from a pluripotent, developmentally plastic state to a committed state during embryogenesis.
The observation that the period of developmental plasticity correlates with the time during which restricted differentiation patterns are being specified in the embryo raises the possibility that the complex transcriptional regulatory networks activated by cell fate specification factors per se
result in the pluripotency → commitment switch. Such gene regulatory networks are known to include positive transcriptional feedback regulatory loops that “lock down” differentiation pathways during specification (e.g. Davidson and Erwin, 2006
; McGhee, 2007
) and the lockdown of one gene regulatory state might be sufficient to prevent the activation of others. If this is the case, then eliminating the function of genes essential for the specification of a cell type might be expected to cause the descendant cells to remain pluripotent. A recent study suggests that this may not be the case at least for pharyngeal cell fates: for example, elimination of the pha-4/FoxA
, critical for pharynx specification, did not result in an extension of the window during which the affected embryonic cells are capable of being reprogrammed (Yuzyuk et al., 2009
). Thus, there may exist global mechanisms controlling pluripotency that are independent of the known cell fate regulatory programs.
Such a global mechanism controlling pluripotency might be expected to reside at the level of changes in chromatin organization. Indeed, Yuzyuk et al. found that, concomitant with the embryonic pluripotent → commitment transition, nuclear chromatin appears to become more condensed, based both on alterations in the morphological appearance of extrachromosomal transgenic elements and on the propinquity of endogenous chromosomal genes detected by DNA in situ hybridization of chromosomes. Thus, chromatin appears to undergo dramatic reorganization as cells lose pluripotency during this transition.
One component that might be expected to direct changes in chromatin organization during the transition from pluripotency to commitment is the polycomb repressor complex, which was first identified in D. melanogaster
based on its role in maintaining differentiation (Lewis, 1978
; Struhl, 1981
), and which has subsequently been shown to be important for pluripotency. In both mouse and human stem cell models (Pasini et al., 2008
), the polycomb complex prevents differentiation of ES cells by repressing genes involved in differentiation and also functions in the stem cell niche in plants (Xu and Shen, 2008
). Members of the polycomb complex have been shown to be indispensable for the self-renewal of neural progenitor cells (Roman-Trufero et al., 2009
) and restrict differentiation potential in neural cell lineages (Hirabayashi et al., 2009
). Studies by Yuzyuk et al. in C. elegans
showed that components of the PRC2 polycomb complex, which methylates histone H3, is not required to maintain developmental plasticity or specification per se
, but is necessary for the switch from pluripotency to commitment.
Transcriptional profiling experiments revealed a number of genes expressed in early C. elegans
embryos that are downregulated during the pluripotency → commitment transition. Mutants lacking MES-2, the PRC2 repressor complex protein E(z), which has also been implicated in repression of HOX gene expression (Ross and Zarkower, 2003
), show prolonged expression of these normally early-specific genes, demonstrating that PRC2 is required for their downregulation. Further, genes associated with ongoing differentiation that are normally expressed late in the transition fail to reach normal expression levels at this time. These findings suggest that mes-2(−)
embryos retain characteristics of early embryos that have not yet committed to particular differentiation pathways. Indeed, the transition from a pluripotent condition into a committed state fails to occur at the normal time in these mutants: cells remain competent to become reprogrammed by heterologous cell fate regulators of muscle and intestinal differentiation beyond the normal window of plasticity (Yuzyuk et al., 2009
). In addition, the mutants do not undergo the same extent of chromatin condensation during the pluripotency → commitment transition, and this apparently less condensed chromatin is associated with higher transcriptional activity based on the presence of phosphoserine2 on the RNA pol II CTD. These findings argue that PC2-directed remodeling of chromatin is responsible for the transition from a plastic, pluripotent state to a committed state of differentiation.