Developmental Biology 268 (2004) 1 – 6 www.elsevier.



How cells dedifferentiate: a lesson from plants
Gideon Grafi *
Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel Received for publication 20 August 2003, revised 9 November 2003, accepted 19 December 2003

Abstract The remarkable regenerative capacity displayed by plants and various vertebrates, such as amphibians, is largely based on the capability of somatic cells to undergo dedifferentiation. In this process, mature cells reverse their state of differentiation and acquire pluripotentiality—a process preceding not only reentry into the cell cycle but also a commitment for cell death or trans- or redifferentiation. Recent studies provide a new perspective on cellular dedifferentiation, establishing chromatin reorganization as its fundamental theme. D 2004 Elsevier Inc. All rights reserved.
Keywords: Cellular dedifferentiation; Regeneration; Pluripotentiality; Cell cycle; Chromatin remodeling; Retinoblastoma; Thrombin; Plants; Amphibians

Introduction It is well established that differentiated plant cells do not lose their developmental potentialities during normal development but retain plasticity, that is, they are capable to dedifferentiate and acquire new fates. It is only when differentiation brings about extreme phenotypic alterations, such as death of the protoplast in tracheary elements (the conducting cells of the xylem) or loss of the nucleus in sieve elements (the conducting cells of the phloem), that plant cells permanently lose developmental potentialities and differentiation is terminal (Steeves and Sussex, 1989). It has long been thought that animal cells once committed to a specific lineage can no longer change their fate and thus become so-called ‘‘terminally differentiated.’’ Recent studies, however, suggest that differentiated animal cells do retain plasticity and can be induced to assume new fates. Such plasticity has been well documented (Graf, 2002; Liu and Rao, 2003; Tosh and Slack, 2002) and is best exemplified in nuclear cloning whereby somatic nuclei transplanted into enucleated oocytes undergo epigenetic reprogramming leading to reestablishment of totipotency (Wilmut et al., 1997). One aspect of cell plasticity is dedifferentiation, a process underlying topical, yet not well-understood issues in biology, such as regeneration, nuclear cloning, establishment of

* Fax: +972-8-934-4181. E-mail address: 0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2003.12.027

new stem cell lineages, as well as carcinogenesis (Brockes and Kumar, 2002; Echeverri and Tanaka, 2002; Odelberg et al., 2002; Sanchez Alvarado, 2000; Sell, 1993; Wilmut et al., 2002). Much of the work on cellular dedifferentiation has dealt with the origin of the amphibian regeneration blastema, a group of ‘unspecialized’ cells from which a lost body part develops. Experiments using x-radiation clearly demonstrated that blastema cells are derived from the limb tissue itself and do not migrate into the limb from other regions of the body (Butler, 1933, 1935). Using tritiated thymidine to follow cell proliferation, Hay and Fischman (1961) showed that blastema cells originated from dedifferentiating internal tissues of the limb including muscle, nerve sheath, and connective tissue. Early works on cellular dedifferentiation emphasized the morphological changes that occur in cells during limb regeneration or when isolated from their original tissue and cultured in vitro. These changes were often interpreted as a sign of dedifferentiation in the sense of assuming a more generalized embryonic state characterized by structural ‘simplicity’ (Bloom, 1937; Thornton, 1938, and references therein). It was argued, however, that cell morphology may not be a reliable measure for ‘true’ dedifferentiation and a more fundamental change in cell identity should be sought (Bloom, 1937; Godman, 1958). Fine examination by electron microscopy was suggested to provide a more definitive criterion as exemplified by changes in the fine structure of muscle fibers during salamander limb regeneration (Hay, 1959). As Hay (1959) stated, however, the ultimate proof for dedifferentiation is the ability of any given cell to differentiate into cell


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Fig. 1. Representation of the plant protoplast system used to study cellular dedifferentiation. Differentiated mesophyll cells respond to the removal of cell wall (cellulase) by undergoing dedifferentiation, thus becoming pluripotent. At this stage, additional signals determine cell fate: auxin and cytokinin induce reentry into S phase and proliferation, auxin by itself may induce redifferentiation (Valente et al., 1998), whereas in the absence of hormonal application, cells die (Zhao et al., 2001).

types other than the one from which it took origin. This could be accomplished only after methodologies for tracking the fate of individual muscle fibers during limb regeneration had been established. Indeed, transplantation of rhodamine dextran- or fluorescent-labeled multinucleated myotubes into regenerating newt limbs resulted in fragmentation into mononucleated cells followed by proliferation (Echeverri et al., 2001; Lo et al., 1993). As elaborated in this review, while dedifferentiation is commonly associated with reentry into the cell cycle, its distinguishing feature is the withdrawal from a given differentiated state into a ‘stem cell’-like state that confers pluripotentiality (Bloom, 1937; Echeverri and Tanaka, 2002; Hay, 1959; Odelberg, 2002; Zhao et al., 2001)—a process preceding not only reentry into the cell cycle but also trans- or redifferentiation or a commitment for cell death (see Fig. 1). This early phase, a fundamental theme in cellular plasticity, has not been sufficiently addressed largely because of lack of a suitable experimental system. Consequently, most biochemical studies related to dedifferentiation in animals focused on the G1 to S transition, ignoring the question of how cells acquire pluripotentiality.

Cellular dedifferentiation: the chromatin viewpoint Cellular dedifferentiation both in plants and animals is characterized by remarkable changes in the pattern of gene expression (Galun, 1981; Jamet et al., 1990; Michalopoulos and DeFrances, 1997), as cells switch from a program that drives the specific function of a somatic cell to a new one directing either reentry into the cell cycle, cell death, or trans- or redifferentiation; the latter refers to the conversion of a differentiated cell type to another without passing through the cell cycle. Thus, the way by which cells remodel their gene expression program is central to understanding the process of cellular dedifferentiation. Since a given gene expression program is governed by the portion of the genome that is transcribed (euchromatin)

versus that which is repressed (heterochromatin), it is likely that for a cell to change its fate, a new balance between euchromatin and heterochromatin must be established. The significance of chromatin structure in switching fate is demonstrated by the conversion of fibroblast mouse cells into new mesenchymal phenotypes (striated muscle cells, adipocytes, and chondrocytes) upon treatment with 5azacytidine (Taylor and Jones, 1979); the latter, after being incorporated into DNA, reduces methylation, which in turn affects chromatin structure and gene expression (Jones and Wolffe, 1999). The basic structural unit of chromatin, the nucleosome, is made up of DNA wrapped around histone octamer containing two copies of each of the four core histone proteins, H2A, H2B, H3, and H4 (reviewed in Kornberg and Lorch, 1999). This basic structure is further organized into higher order chromatin structure by the aid of multiple proteins or protein complexes (reviewed in Grewal and Moazed, 2003). Dynamic changes in chromatin structure are directly influenced by modifications of the DNA (e.g., by methylation) as well as by posttranslational modifications of histone amino terminal tails. Within histone tails, there are specific amino acids (arginine, lysine, and serine) that undergo a number of posttranslational modifications, including acetylation, methylation, and phosphorylation (van Holde, 1989; Wolffe, 1992), thus generating a ‘code’ for the recruitment of protein complexes that affect chromatin structure and function (reviewed by Grewal and Moazed, 2003; Jenuwein and Allis, 2001; Zhang and Reinberg, 2001). For example, methylation of histone H3 at lysine 9 by histone H3-K9 methyltransferase Suv39h1 (Rea et al., 2000) generates a ‘code’ for the recruitment of heterochromatin protein 1 (HP1) (Bannister et al., 2001; Lachner et al., 2001), a chromodomain protein that is involved in the assembly of heterochromatin and gene silencing (Cavalli and Paro, 1998). Conversely, modification of histones by acetylation is often associated with ‘open’ chromatin configuration and gene transcription (reviewed by Eberharter and Becker, 2002).

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The linkage between cellular dedifferentiation and chromatin reorganization has been recently demonstrated in studies using the plant protoplast system (Avivi et al., in press; Williams et al., 2003; Zhao et al., 2001). Plant protoplasts (plant cells devoid of cell walls) appear to provide a valuable experimental tool for studying how cells acquire pluripotentiality. The fully differentiated, nondividing mesophyll cells can be separated from their original tissue by cell-wall-degrading enzymes generating a large population of protoplasts that are not yet committed to any specific fate but have acquired pluripotentiality; their subsequent fate depends on the type of stimulus applied (Fig. 1) (Zhao et al., 2001). Only upon application of phytohormones (auxin and cytokinin) can these protoplasts reenter the cell cycle, proliferate, and form calli (masses of dividing cells) from which shoots and roots can be regenerated to form the entire fertile plant (Takebe et al., 1971). Hence, the transition of a differentiated mesophyll cell into the cell cycle is resolved into two distinct phases: acquisition of pluripotentiality (dedifferentiation), followed by a signaldependent reentry into S phase (Fig. 1). As demonstrated in the protoplast system, dedifferentiation precedes not only reentry into S phase but also redifferentiation or cell death (Fig. 1). By monitoring chromatin structure, two distinct phases of chromatin decondensation were evident: an early phase that occurs during acquisition of pluripotentiality followed by a second phase that precedes reentry into S phase (Zhao et al., 2001). Acquisition of pluripotentiality was accompanied by posttranslational modifications of histone H3, redistribution of heterochromatin protein 1, as well as by disruption of the nucleolar domain, which was accompanied by chromatin condensation of the 18S ribosomal DNA (Fig. 2) (Williams et al., 2003). Moreover, chromatin decondensation appears to be subdomain-specif-

Fig. 2. Reorganization of the nucleolar domain during acquisition of pluripotentiality in Nicotiana tabacum. Fluorescence in situ hybridization (FISH) analysis demonstrating the dispersed chromatin configuration of the 18S rDNA gene cluster (green) in differentiated leaf cells versus condensed configuration in pluripotent protoplasts. Note the disruption of the nucleolar domain in pluripotent protoplasts revealed with DAPI staining (blue). Scale bar = 10 Am (from Williams et al., 2003).

ic, leading to activation of silent genes such as VIP1 and NO APICAL MERISTEM (NAM)-like genes (Avivi et al., in press). While VIP1 encodes a VirE2-interacting protein that is required for Agrobacterium tumorigenecity (Tzfira et al., 2001), NAM domain proteins are assumed to be involved in establishing new stem cell lineages in plants (Duval et al., 2002; Souer et al., 1996). Petunia embryos carrying a mutated NAM gene failed to develop a shoot apical meristem (Souer et al., 1996), the zone at the tip of a shoot acting as a self-renewing source of pluripotent stem cells from which all plant tissues are formed. Thus, NAM may confer pluripotentiality in plants similarly to Oct3/4 in animals (Pan et al., 2002). Notably, Oct4 was shown to be activated in mammalian somatic nuclei upon transplantation into Xenopus oocytes in a cell cycle-independent manner (Byrne et al., 2003), supporting the notion of pluripotentiality preceding a switch in cell fate. Chromatin reorganization has been reported in a variety of dedifferentiating eukaryotic cells. When chicken erythrocyte nuclei were incubated in Xenopus egg cell-free extract, they displayed two distinct phases of chromatin decondensation before reactivation of DNA synthesis (Blank et al., 1992). By analogy to plant protoplasts, the first phase of chromatin decondensation in chicken erythrocyte nuclei may represent nuclear reprogramming leading to acquisition of pluripotentiality, while the second phase is related to acquisition of a new fate, namely, reentry into the cell cycle. Similar to plant protoplasts, somatic nuclei transplanted into Xenopus egg extract showed rapid disassembly of nucleoli (Gonda et al., 2003; Kikyo et al., 2000), suggesting that structural reorganization of nucleoli is a common feature of dedifferentiating cells both in plants and animals. Chromatin reorganization was reported in frog red blood cells that were induced to reenter the cell cycle; these cells displayed a unique cytofluorometry histogram before entry into S phase, which was interpreted as chromatin decondensation (Chiabrera et al., 1979). Also, chromatin prepared from regenerating Owenia fusiformis (polychaete annelid) 12 h after amputation was more accessible than that from intact animals to nucleases, suggesting decondensation of chromatin (Fontes et al., 1980). Hence, chromatin reorganization appears to be a fundamental theme both for cellular dedifferentiation and for reentry into the cell cycle, providing the means for resetting the gene expression program. The cascade of events leading differentiated animal cells to switch fate is not clear. Myotubes, long considered terminally differentiated, were often used to study how cells change fate (Echeverri and Tanaka, 2002; Odelberg, 2002; Tosh and Slack, 2002). Switching fate in myotubes was described in numerous studies; depending on the signal provided, myotubes can be induced either to reenter the cell cycle and proliferate, die, or transdifferentiate directly into multiple cell types (Endo and Nadal-Ginard, 1998; Odelberg, 2000; Tosh and Slack, 2002). Taking a lesson from plants, I argue that for a myotube, as for any other cell,


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to change fate it must first go through a preparatory stage that confers pluripotentiality. As outlined below, reassessment of published data provides support for this idea. Dedifferentiation of myotubes was often correlated with inactivation of the retinoblastoma protein (pRb), a master regulator of cell cycle, differentiation, and apoptosis (reviewed in Herwig and Strauss, 1997). This was demonstrated in mouse myotubes lacking pRb, which unlike their wild-type counterparts could be induced by serum to reenter S phase (Schneider et al., 1994). Likewise, myotubes derived from vertebrates that are endowed with high regenerative capabilities (such as newts) reenter S phase upon stimulation with serum, a process that requires inactivation of pRb by phosphorylation (Tanaka et al., 1997). Furthermore, overexpression of viral oncogenes, reconstitution of a cyclin D/CDK complex (both of which lead to pRb inactivation), and conditional knockout of pRb enabled differentiated cells cultured in serum to reenter S phase (Endo and Nadal-Ginard, 1998; Latella et al., 2001; Sage et al., 2003; Tiainen et al., 1996). Apparently, the mere deficiency or inactivation of pRb, while essential, is insufficient to reactivate S phase (Mal et al., 2000; Schneider et al., 1994). It appears that in the experiments mentioned above, additional activities residing within the serum allow differentiated cells to become responsive to the loss of pRb by reentry into the cell cycle. As shown for the plant protoplast system, I suggest that this ‘responsiveness’ triggered by the serum is achieved through acquisition of pluripotentiality. Indeed, myotubes expressing the oncogene SV40 large T antigen were induced by serum either to reenter the cell cycle, undergo apoptosis, or form giant or pulverized nuclei (Endo and Nadal-Ginard, 1998), thus providing evidence for ‘true’ dedifferentiation. Interestingly, this serum activity was not mediated by growth factors but by a factor(s) that was activated by thrombin proteolysis (Imokawa and Brockes, 2003; Simon and Brockes, 2002; Tanaka and Brockes, 1998; Tanaka et al., 1997). If indeed, as discussed previously, chromatin reorganization underlies dedifferentiation and precedes switch in cell fate, what could drive the structural reorganization of chromatin in these experiments? The answer may lie in chromatin remodeling activity triggered by the serum via thrombin proteolysis or by the experimental procedure itself (e.g., viral infection). Besides its involvement in blood coagulation, thrombin was found to function as a potent activator and growth factor of vascular smooth muscle cells. In these cells, thrombin induces hypernuclear acetylation mediated by the MAP kinase pathway, at least partly by stimulating histone acetyltransferase (HAT) activity (Kawahara et al., 1999). Increased HAT activity is often associated with chromatin decondensation and gene transcription (reviewed by Eberharter and Becker, 2002), thus linking thrombin signaling with chromatin remodeling activity. Furthermore, chromatin remodeling-induced dedifferentiation may be brought about by viral infection often used to deliver genes into cells. This is illustrated in cells infected with herpes

simplex virus 1 (HSV-1) where shortly after infection, these cells displayed extensive reorganization of chromatin, and similarly to dedifferentiating cells, they also displayed disruption of the nucleolar structure (Monier et al., 2000). Also, the Xenopus egg extract, often used to study reactivation of S phase in somatic nuclei, was shown to contain the chromatin-remodeling nucleosomal adenosine triphosphatase (ATPase) ISWI, which is required for transplanted somatic nuclei to reenter S phase (Kikyo et al., 2000). ISWI, a member of the SWI2/SNF2 superfamily, is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin (Varga-Weisz and Becker, 1998; Vignali et al., 2000).

Fig. 3. A proposed model for dedifferentiation. This model proposes that cellular dedifferentiation, that is, acquisition of pluripotentiality, is driven by chromatin remodeling often associated with reorganization of the nucleolar domain. Chromatin remodeling leads to activation of pluripotent genes such as NAM in plants and Oct3/4 in animals, thus establishing pluripotentiality. Such remodeling could be brought about by various stimuli such as thrombin proteolysis, viral infection, chromatin remodeling factors in the Xenopus egg extract, as well as by the removal of cell walls in plants. Inactivation of pRb by oncoproteins such as E1A or by phosphorylation may also be involved in chromatin remodeling associated with pluripotentiality but more likely with chromatin remodeling associated with reentry into the cell cycle. It is proposed that pRb inactivation, Msx1 overexpression, as well as various growth factors act after pluripotentiality had been established to induce new cell fates, that is, cell cycle, cell death, or redifferentiation. HAT, histone acetyltransferase; ISWI, a member of the SWI2/SNF2 chromatin-remodeling adenosine triphosphatase; HP1, heterochromatin protein 1.

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Concluding remarks Based on work done in plants and animals, a model is proposed (Fig. 3) depicting cellular dedifferentiation from the perspective of chromatin structure. Cellular dedifferentiation is driven by chromatin reorganization leading to activation of pluripotent genes such as NAM in plants and Oct3/4 in animals, thus establishing pluripotentiality. Chromatin remodeling-induced pluripotentiality may be brought about by a wide variety of stimuli such as thrombin activity in the serum, viral infection, removal of cell walls in plants, or chromatin remodeling factors residing in Xenopus egg extract. Inactivation of pRb may also lead to chromatin remodeling associated with pluripotentiality but more likely with chromatin remodeling associated with reentry into S phase (Ghosh and Harter, 2003; Ogawa et al., 2002). It is proposed that pRb inactivation, Msx1 overexpression (Odelberg et al., 2000), as well as various growth factors could induce switch in cell fate only after the state of pluripotentiality had been attained. It would be useful to explore animal systems where the process of dedifferentiation can be easily resolved. Transplantation of somatic nuclei into Xenopus egg extract or Xenopus oocytes may serve this purpose (Blank et al., 1992; Byrne et al., 2003). In this system, it should be feasible to study the molecular basis for chromatin reorganization and its significance for acquisition of pluripotentiality. A particular consideration should be given to the nucleolus reorganization (a hallmark event of pluripotentiality?) and to activation of pluripotent genes such as NAM in plants (Avivi et al., in press) and Oct3/4 in animals (Byrne et al., 2003). Remodeled chromatin subdomains are expected to possess genes whose products are involved in acquisition of pluripotentiality and/or the ensuing fate of the cell. These subdomains may be identified using as markers either upregulated genes (e.g., by DNA chip technology) or DNA sequences undergoing changes in methylation pattern (Avivi et al., in press). Exploiting the dedifferentiation process to obtain new stem cell lineages holds a great promise for regenerative medicine, particularly in view of the debate over the use of human embryonic stem cells in research and the ethical concerns it raises. The use of nonanimal experimental systems (such as plant protoplasts) or in vitro systems (such as nuclear transplantation into Xenopus egg extract) could greatly enhance stem cell research. Insights gained through these systems might one day lead to the development of methodologies that could aid in reprogramming human cells for clinical applications.

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Acknowledgments I thank Y. Avivi for critical reading of the manuscript, E. Galun and S. Lev-Yadun for their helpful comments, and an anonymous reviewer for his thoughtful comments. Research


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