Molecular Reproduction & Development 77:837–855 (2010)
Cell Plasticity in Homeostasis and Regeneration
BRIGITTE GALLIOT,* AND LUIZA GHILA
Faculty of Sciences, Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland
Over the past decades, genetic analyses performed in vertebrate and invertebrate organisms deciphered numerous cellular and molecular mechanisms deployed The ability of an organism to during sexual development and identi，ed genetic circuitries largely shared among regenerate depends on its bilaterians. In contrast, the functional analysis of the mechanisms that support capacity to access a source of regenerative processes in species randomly scattered among the animal kingdom, stem cells and/or to reprogram were limited by the lack of genetic tools. Consequently, unifying principles explaining differentiated cells how stress and injury can lead to the reactivation of a complete developmental program with restoration of original shape and function remained beyond reach of understanding. Recent data on cell plasticity suggest that beside the classical developmental approach, the analysis of homeostasis and asexual reproduction in adult organisms provides novel entry points to dissect the regenerative potential of a * Corresponding author: given species, a given organ or a given tissue. As a clue, both tissue homeostasis and Sciences III, regeneration dynamics rely on the availability of stem cells and/or on the plasticity of 4 Bd d’Yvoy, CH-1211 differentiated cells to replenish the missing structure. The freshwater Hydra polyp Geneva 4, Switzerland. provides us with a unique model system to study the intricate relationships between E-mail: firstname.lastname@example.org the mechanisms that regulate the maintenance of homeostasis, even in extreme conditions (starvation and overfeeding) and the reactivation of developmental pro- grams after bisection or during budding. Interestingly head regeneration in Hydra can follow several routes according to the level of amputation, suggesting that indeed the homeostatic background dramatically influences the route taken to bridge injury and regeneration.
Mol. Reprod. Dev. 77: 837–855, 2010. ß 2010 Wiley-Liss, Inc. Published online in 2 July 2010 Wiley Online Library (wileyonlinelibrary.com). Received 10 December 2009; Accepted 1 May 2010 DOI 10.1002/mrd.21206
meaning that the differentiated cells can undergo cell growth INTRODUCTION TO ADULT DEVELOPMENTAL
but no proliferation during adulthood. The nematodes that BIOLOGY
keep their number of somatic cells constant in adulthood, A wide range of distinct biological processes contribute to provide the best example; similarly, in Drosophila all somatic the preservation of the anatomical form and functionality in adult tissues are post-mitotic except the gut. This drastic adult animal organisms; these processes are acting at regulation of adult cell number generally impedes adult different levels, such as metabolism that affects the whole plasticity, which is required for homeostatic or regenerative organism, cell turnover of organs and tissues, autophagy of mechanisms. However, in most metazoan species, the main speci，c cell types, DNA repair at the nuclear level (Rando, way to protect adult organisms from physiological dysfunc- 2006). As human beings, we often consider that a high cell tions involves the removal and replacement of old or dam- turnover is an obligatory rule to maintain the integrity of adult organisms. However, this is certainly not systematically observed across animal phyla as several species with short Abbreviations: AEC, apical epithelial cap; ASC, adult stem cell; GRN, gene lifespan can be strictly post-mitotic after development, regulatory network.
ß 2010 WILEY-LISS, INC.
Molecular Reproduction & Development ALLIOT AND GHILA G
aged differentiated cells. This ongoing physiological force and to maintain the change after this force has ceased replacement process is named cell turnover. The adult stem to act’’ (from Littre French dictionary, translated by Will et al., cells (ASCs) play a key role in this turnover, although limited 2008). At the ，rst look, this de，nition apparently applies quite to the organ or the tissue where they reside (Wagers and well to the regenerative process, however, the usage of the Weissman, 2004; Ohlstein and Spradling, 2006; Blanpain word plasticity in biology is much broader, focusing on the et al., 2007). As a classical scenario, ASCs divide through ability of living organisms to adapt to constraints by changing asymmetric division, with one of the daughter cells keeping their organization at a speci，c level, for example, evolution- the ‘‘stemness’’ status (self-renewal) whereas the second ary, developmental, phenotypic, synaptic, cellular, and mo- one, no longer a stem cell, undergoes a series of cell division, lecular. As a consequence, the word ‘‘plasticity’’ should
providing a transient amplifying stock that will subsequently never be used alone but always be speci，ed by the level
commit to one or a series of differentiated fates (Raff, 2003). where it applies (Pomerantz and Blau, 2004). Some As a consequence three competitive processes regulate scientists even proposed to apply to the concept of plasticity homeostasis: cell death, cell proliferation, and cell differen- in biological systems a more ‘‘engineer-oriented’’ usage,
tiation. The study of their crosstalk in Drosophila imaginal restricting it to the contexts where lasting structural reorga- discs showed how a coordinated cell–cell signaling tightly nization, that is, modi，cations of the material structure of regulates this competition in a given tissue (Moreno and the system (interface, connectivity network, constitutive Basler, 2004). In mammalian tissues, cell turnover occurs in elements), are indeed proven, leaving out of plasticity the epidermis, intestine, lung, blood, bone marrow, thymus, effects of variability, flexibility, systematic variations, and testis, uterus, and mammary gland with large variations in vicarious (substituted) processes as these effects rather the rate of cell turnover, from few days for the intestinal result from ‘‘operational’’ than structural changes (Will
epithelium up to several months for the lung epithelium et al., 2008). We selected here few examples to discuss (Blanpain et al., 2007). In other organs (brain, heart, pan- this view, certainly more rigorous or at least less metaphoric creas, kidney, cornea, etc.), the physiological cell turnover is (following the words of Will et al., 2008) but as we will see, likely limited and/or very slow, making dif，cult the in vivo dif，cult to apply in some contexts.
monitoring of the respective behaviors of stem cells and Evolutionary plasticity is certainly the best example of dying cells. plasticity with structural changes leading to lasting changes. Similar to cell turnover, tissue repair also allows tissue The combination of genomic, genetic, and developmental replacement but requires the damage-induced activation of approaches over the past 20 years have de，nitively proven
programs that monitor cell proliferation and cell differentia- that variations in the genomic organization of the Hox gene tion. Finally, regeneration of anatomical structures like clusters obviously lead to genetic reprogramming during appendages, represent an even more complex process with development and to species-speci，c modi，cations of the
formation of a transient proliferative structure, the blastema, body plan (Duboule, 2007). Developmental plasticity that and activation of a developmental program that leads to was identi，ed ，rst in sea urchin embryos by Driesch in 1892, restoration of original shape and function (Brockes and and later in vertebrate embryos, refers to the embryonic Kumar, 2005). Both tissue repair and regeneration that potential for regulation as the embryonic cells at early stages affect different tissue types and require cell replacement on have the ability to change their fate to compensate for cell a large-scale, are triggered by nonspeci，c and usually loss (Driesch, 1900). This potential, which accounts for the exogenous damage, whereas cell turnover is a process that occurrence of homozygous twins, is transient but can still be is endogenously initiated and restricted to a fraction of cells observed at later stages in more specialized tissues as limb (Pellettieri and Sanchez Alvarado, 2007). buds (Summerbell, 1981) or neural crest cells (Vaglia and Nevertheless one can intuitively perceive a progression Hall, 1999). Developmental plasticity, more recently named from basic tissue self-renewal to tissue repair, reached by transfating (Keleher and Stent, 1990), requires the activation some but not all organs, to regeneration, accessed by a of the gene regulatory network (GRN) that corresponds to
the new cell fate. Interestingly, in sea urchin embryo this ‘‘happy few’’ elite of organs or structures. This view suggests
a possible continuum between the processes that regulate activation apparently depends on inputs that are distinct each step, even though their complexity is supposed to during normal and regulative developments (Ettensohn gradually increase. To challenge the solidity of this view, et al., 2007). If con，rmed as a general rule, this would mean we review some results recently obtained in the paradig- that context-speci，c signals sensed at the ‘‘interface’’ of the
matic Hydra model system. But before considering the system induce long-lasting structural reorganizations of the different forms of plasticity deployed in Hydra, we will ，rst developing organism.
discuss the origin and the current meaning of the concept of Phenotypic plasticity is ‘‘the property of a given genotype plasticity. Indeed, this concept is widely used by biologists to produce different phenotypes in response to distinct from different ，elds, but sometimes covering quite distinct environmental conditions’’ (Metcalf, 1906), with the ，rst
meanings. study of adaptive phenotypic plasticity described in the
crustacean Daphnia. However, the different phenotypes
might reveal an intrinsic ‘‘repertoire of competences’’ that The Ambiguities of the Concept of ‘‘Plasticity’’ need no structural changes to be expressed (Will et al., The word ‘‘plasticity’’ (from Latin plasticus or Greek 2008). In the same year, 1906, the term neuroplasticity was plastikos, ability to mold) refers to the ‘‘capacity of distortable proposed by Ernesto Lugaro, a psychiatrist, who referred to bodies to change their shape under the action of an external the changes in neural activity during psychic maturation,
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cell states before and after transdifferentiation (Wagers and learning processes, or post-damage recovery (Berlucchi,
2002). During the ，rst half of the 1900s, the concept of brain Weissman, 2004; Slack, 2007). In fact, the most compelling plasticity was rejected by the scienti，c community, as it was evidence is provided by the transient co-expression of unanimously accepted that the fully developed brain markers of the two differentiated cell states (Schmid and reached stability at adulthood, each region of the brain Alder, 1984).
performing speci，c function(s) that could not be modi，ed. More recently, it was possible to induce transdifferentia- In the 1960s, this view started to be challenged by experi- tion by overexpressing one or several cell-speci，c transcrip-
ments proving activity-dependent brain plasticity (Bennett tion factors that suf，ce to convert one cell type to another et al., 1964; Bach-y-Rita et al., 1969). Synaptic plasticity, the (Slack, 2007; Eberhard and Tosh, 2008; Zhou et al., 2008). capability for a neuron to modify on the long term its Indeed nuclear reprogramming plays an essential role in electrophysiological activity according to the stimuli it had cellular plasticity and developmental biologists actually received, was ，rst studied in the mollusk Aplysia (Bruner and provided the ，rst experimental evidence of this event: they Tauc, 1965; Kandel and Tauc, 1965). The choice of this showed that nuclei isolated from mature somatic cells model system was instrumental to establish the importance and transplanted into enucleated Xenopus oocytes, could of plasticity in the learning and memory processes as per- reprogram and orchestrate the development of a frog sistent modi，cations of the activity of the genetic circuitry are (Gurdon et al., 1958). This surprising ，nding meant that
required to sustain changes in neurophysiological activity nuclei of terminally differentiated cells can become totipo- (Barco et al., 2006). tent. Forty years later, the cloning of the sheep Dolly, also Cellular plasticity is directly related to the questions obtained by nuclear transfer from an adult somatic tissue, addressed in this review, that is, what conditions of tissue the mammary gland, con，rmed this major ，nding in
homeostasis support a regenerative response. For this mammals (Wilmut et al., 1997). Actually, even nuclei from reason, we will discuss here only the cellular plasticity of post-mitotic neurons can be reprogrammed to drive the somatic cells (Fig. 1). As a ，rst but rather rare strategy complete development of mice (Eggan et al., 2004). differentiated cells can re-enter the cell cycle after injury, Finally, since 2006 reprogramming of mature somatic as exempli，ed by hepatocytes in mammals (Rabes et al., cells can be pushed to the point where adult differentiated 1976). More frequently adult differentiated cells actually cells directly reach an embryonic-like stemness thanks to dedifferentiate upon injury before entering an active cycling the co-expression of de，ned transcription factors without phase to form a blastema (see below). But cells can also using oocytes. Such cells, named induced pluripotent stem undergo metaplasia, that is, phenotypically convert from one cells (iPSC), were obtained so far from ，broblasts
cell or tissue type into another, a process well known by (Takahashi and Yamanaka, 2006; Takahashi et al., pathologists, which actually covers a variety of processes. 2007), lymphocytes (Hanna et al., 2008), keratinocytes Among those, transdifferentiation is de，ned by the fact (Aasen et al., 2008), cord blood cells (Haase et al., 2009), that stably differentiated cells irreversibly change their fate, smooth muscle cells (Lee et al., 2010). Whatever the proce- that is, reprogram by acquiring a novel differentiated status dure, reprogramming relies on epigenetic changes (Loh with a speci，c molecular signature (Okada, 1991; Eguchi et al., 2008; Hochedlinger and Plath, 2009), which certainly and Kodama, 1993). During that process, the cells may or correspond to ‘‘material’’ changes although not necessarily may not traverse the cell cycle. Similarly cell fusion that, as ‘‘structural’’ changes.
transdifferentiation is also increased upon injury, might lead
to reprogramming when two distinct cell types fuse (Chiu and
Two Emerging Model Systems for Investigating Blau, 1984; Pomerantz and Blau, 2004). Obtaining the Homeostasis and Regeneration experimental proofs of transdifferentiation is often dif，cult,
Two historical invertebrate model systems, Hydra and but at least morphological and molecular criteria as well as
cell lineage relationships should clearly characterize the two planarians, were long recognized to be suitable for investi-
gating the mechanisms supporting tissue homeostasis, ac-
tive maintenance of patterning in adulthood as well as
complex cellular reorganization to regenerate after injury.
The freshwater polyp Hydra belongs to Cnidaria, a sister
phylum to bilaterians, and the flatworm planaria that belongs to Lophotrochozoa (see their respective phylogenetic posi- tions in Fig. 2) actually share ，ve cellular and developmental features: (1) An intense and continuous tissue replacement in adult- hood due to a stock of mitotically active stem cells, unique in case of planarians (the neoblasts), and three- fold in case of Hydra (the ectodermal epithelial stem cells, the endodermal epithelial stem cells, and the interstitial stem cells).
(2) A stock of adult pluripotent stem cells that produce germ Figure 1. The different forms of cellular plasticity that can be observed cells and somatic cells throughout the life of the animals or induced in differentiated somatic cells.
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Figure 2. Wide distribution of the regenerative potentials across the animal kingdom. Phylogenetic tree of the animal species exhibiting a
regenerative potential after injury.
(the interstitial stem cells in Hydra; the neoblasts in variations in the feeding diet, and second after bisection,
planarians), a situation quite unique among most animal when the animal survives the amputation stress and regen-
phyla where germ cells usually segregate during early erates the missing part. Given that most gene families that
embryonic development. control cellular and developmental behaviors are present (3) An ef，cient asexual reproduction mechanism, through and highly conserved in cnidarians (Putnam et al., 2007;
budding in Hydra and ，ssion in planaria. Chapman et al., 2010), these forms of plasticity are likely not (4) The amazing property to regenerate almost any missing exotic and we will discuss the correspondences between
part of the body after injury. these changes and those observed in various bilaterian (5) An apparent lack of aging, at least when the animals do model systems.
not enter the sexual cycle (Martinez, 1998; Yoshida
et al., 2006; Pearson and Sanchez Alvarado, 2008).
TISSUE PLASTICITY IN HOMEOSTASIS However, Hydra and planarians are not genetically trac- The Hydra body wall comprises two layers, ectodermal table. The recent development of genomic, molecular and
cellular tools promoted their emergence as modern model and endodermal, that together contain about a dozen cell systems where the mechanisms of homeostasis and regen- types. These cells derive from three distinct stem cell eration can now be investigated thanks to RNA interference populations, ectodermal myoepithelial, endodermal myoe- (RNAi) gene knocked down and transgenesis (see in Red- pithelial, and interstitial cells (Dubel et al., 1987; Bode, 1996; dien and Sanchez Alvarado, 2004; Galliot et al., 2006; Steele, 2002; Galliot et al., 2006). The spatial distribution of Bosch, 2007; Bottger and Alexandrova, 2007; Salo et al., stem cells, progenitors, and differentiated cells along the 2009). Homeostasis in planarians was recently reviewed polyp occurs as a consequence of the continuous division of in length (Pellettieri and Sanchez Alvarado, 2007; stem cells in the body column joined to the active migration Handberg-Thorsager et al., 2008; Rossi et al., 2008) and or the passive displacement of the committed/precursor we will report here about the distinct forms of plasticity cells in either apical or basal directions (Fig. 3A). According that take place in adult Hydra polyps, ，rst in response to to the cell types, the cells terminally differentiate during their
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Figure 3. Cellular and morphological variations induced by the feeding diet in Hydra. A: The homeostasis of the Hydra adult polyp relies on the dynamic equilibrium between cell gain and cell loss. Cell proliferation takes place in the body column whereas cells differentiate when they migrate or get displaced to the apical and basal poles, respectively, the head region with tentacles and hypostome (dome surrounding the mouth opening) and the basal disc. Subsequently, these cells get sloughed off from the extremities and are replaced by the continuous in！ux of younger cells. B: The number of cells in a polyp is directly in！uenced by the feeding diet. Here, the cell number by Hydra was plotted against the number of artemia given in the daily feeding (data taken from Bode et al., 1977). For each feeding period a two-degree polynomial function was calculated to show the tendency of cell number changes. C: Morphogenesis in Hydra is directly in！uenced by the feeding diet. Upon starvation the animals stop budding and rapidly activate autophagy to survive, reducing their size but keeping intact their morphology (red left panel). In steady-state condition, the animal size is stable and asexual reproduction, that is, budding, takes place with new buds forming every 2 or 3 days (middle blue panel). In overfeeding conditions (green right panel), homeostasis is not maintained as heteromorphosis (bizarre morphologi- cal changes) precedes the animal death (Otto and Campbell, 1977). Note that in all conditions, bisection triggers regeneration.
move or at their ，nal position. Hence, the extremities, that is, Budding and Autophagy, Two Ways to Maintain the tentacles, the hypostome (dome apex surrounding the Fitness in Hydra
mouth opening) and the basal disk, are made up of terminally In Hydra, the homeostasis mechanisms tightly link cell differentiated cells that are continuously sloughed off. This renewal to an active maintenance of shape and ，tness; this
permanent source of stem cells in the Hydra body column is best illustrated by the adaptation of Hydra to feeding likely confers its unique cellular plasticity among multi- conditions, when the animal regulates its steady state by cellular adult organisms. growing and budding with regular feeding. Upon starvation,
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the steady state is no longer maintained and the animals atrophy (Shostak, 1974). Indeed in the laboratory where it is relatively easy to control the diet of a given population as Hydra are fed with freshly hatched Artemia nauplii, Hydra reduces its size but maintains its shape, ，tness, and regen- erative potential over long periods of starvation (up to 4 weeks). Therefore, numerous studies investigated the mod- ulations of homeostasis in response to nutrient abundance, showing that the feeding diet dramatically influence the morphogenetic processes, namely the budding rate and the
maintenance of patterning, as well as the cycling of the
epithelial cells (Bode et al., 1977; Otto and Campbell,
1977). Three distinct responses were characterized accord-
ing to the level of feeding (Figs. 2C, 3B). In well-fed animals
(3–24 artemia per day), the cell production exceeds tissue growth rate, and the cellular ‘‘surplus’’ is ‘‘eliminated’’ through asexual reproduction—a fast process, which causes the growth of a bud on the parental body, which itself does not grow. This budding process results in doubling the animal number each 2–4 days; the distinct cell popula- tions composing the tissue mass indeed increase but their Figure 4. Cellular and developmental responses to starvation in relative proportions remain stable (David and Campbell, Hydra. Apoptosis is observed after 2 days of starvation, affecting less 1972; Bode et al., 1973). than 2% of cells of the interstitial cell lineage even after a long period of By contrast in over-fed animals (over 25 artemia per day), starvation. In contrast autophagy progressively affects all epithelial both cell proliferation and budding are increased (Bode et al., cells, reaching a plateau level after 11 days. These two cellular responses immediately vanish when animals receive nutrients. Cell 1977; Otto and Campbell, 1977) and the tissue mass ex- proliferation (not represented here) is not signi，cantly affected upon ceeds the loss of tissue caused by budding. Considering that starvation (Bosch and David, 1984). At the developmental level, the tissue loss at the base and at the tentacles is compara- budding requires a regular feeding whereas regeneration does not. tively low, the rate of budding predominantly regulates both Indeed head regeneration is only slightly delayed in 17-days-starved tissue loss and the length of the parent’s body. Some reports animals when compared to daily fed animals (regeneration was not tested after periods of starvation longer than 17 days; Chera et al., actually suggest differences between the different Hydra 2009a). species: the Japanese species H. magnipapillata elongates its body column while the head remains at almost constant size (Kroiher, 1999), whereas the European species H. apoptosis is likely not suf，cient to provide a long-standing vulgaris maintains its proportions (Muller, 1995). However,
the steady state is never reached in overfed animals, energy source. More recently, autophagy was detected which will eventually undergo heteromorphosis (bizarre when feeding is stopped, ，rst in the ectodermal, later on in morphological changes) and die (Bode et al., 1977). the endodermal myoepithelial cells (Buzgariu et al., Finally at low feeding level or under starvation conditions 2008; Chera et al., 2009a). But in contrast to apoptosis (0–1 artemia per day), Hydra polyps rapidly stop budding that remains constant during starvation, autophagy pro- and progressively decrease their size to about half with no gressively affects all epithelial cells, providing a source of alteration of their body shape or their ，tness (Fig. 3C). nutrients over long periods of starvation (Fig. 4). Upon Surprisingly, the relative sizes of the different cell popula- feeding resumption, the animals immediately stop auto- tions, as well as the total number of cells per animal, remain phagy, start to re-grow, and recover their size and their almost constant (Bode et al., 1973). In fact, an imbalance ability to bud in several days. Thus, the data currently between the decrease in polyp size and the cell cycle length available suggest that Hydra adapts to low feeding diet was observed, as cell proliferation initially remains roughly through two distinct cellular mechanisms, autophagy for constant, leading to the overproduction of 10% cells per day energy support and apoptosis for cell number.
(Bosch and David, 1984). This apparent contradiction was
explained when Bosch and David found that apoptosis is
Autophagy in Hydra Leads to Cell Death When actually rapidly induced upon starvation, in about 2 days
Derepressed (Fig. 4). As a consequence the supernumerary cells
A second form of autophagy was actually discovered by produced by cell proliferation during starvation become
pure serendipidity when Kazal1, a Serine Protease INhibitor apoptotic and are engulfed by the neighboring epithelial
Kazal-type (SPINK) gene, was silenced by repeatedly feed- cells, providing a regulatory mechanism for keeping more
ing Hydra with dsRNAs. Progressively an excessive auto- or less stable the cell number (Bosch and David, 1984;
phagy was observed in the endodermal cells of these intact Bottger and Alexandrova, 2007; Pauly et al., 2007).
However, the apoptotic process that affects less than 2% Kazal1(RNAi) Hydra; large autophagosomes formed in the
digestive cells, progressively fusing and leading to cell of the cells, remains stable over the starvation process
(Bosch and David, 1984; Chera et al., 2009a). Therefore, shrinkage and cell death in several days (Chera et al.,
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ELL PLASTICITY IN HOMEOSTASIS AND REGENERATION C
one major aspect of regeneration, which is the regulation 2006). As previously mentioned homeostasis and budding
are tightly linked in Hydra and the ，rst consequence of this and function of cell migration towards the wound. autophagy phenotype was to prevent budding and then to
slowly induce animal death. These data indicate that the Wound Healing in Regenerative and level of autophagy needs to be tightly controlled in steady- Nonregenerative Contexts state homeostasis. The recovery of tissue integrity in response to environ- Interestingly, a de，cit of Kazal-type protein activity also /mental stress, injuries or diseases, encompassed by the leads to excessive autophagy in mammals. In the Spink3 names tissue repair and regeneration, requires in multicel- newborns, autophagosomes rapidly invades the exocrine lular organisms the activation of the wound healing process. pancreatic cells and the surrounding digestive cells Wound healing plays a decisive role in survival by rapidly (Ohmuraya et al., 2005). As in Hydra, these mice never covering the wound with an epithelial layer that secures body gain weight and die in a couple of weeks. Therefore, in mice integrity and avoids tissue loss and infections. However in as in Hydra the Spink3 and Kazal1 proteins that are mammals, aging dramatically affects wound healing: em- produced by similar exocrine cells, that is, the zymogen bryos and fetuses are able to heal rapidly, ef，ciently, and pancreatic cells and the gland cells respectively, appear to without scarring, whereas in adults wound healing is often play a similar function, that is, to protect the cells that imperfect as in the case of the skin where it is limited to produce the digestive enzymes and the digestive cells from scarring (Redd et al., 2004). Indeed scarring and wound self-digestion. This is the ，rst example where a similar healing are not identical as that latter process requires the pathological cellular process regulated by related gene unsilencing of repair genes through epigenetic reprogram- families can be traced from cnidarians to mammals. ming in the wound epidermis (Shaw and Martin, 2009). An Autophagy in eukaryotic cells involves the sequestration additional level of complexity was observed in vertebrates and degradation of cytoplasmic organelles via the lysosomal that regenerate appendages as the wound epidermis that pathway, as such it participates in the maintenance of covers the amputation plane thickens to form a structure cellular homeostasis by generating nutrients but also by named the apical epithelial cap (AEC), which is an equiva- preventing the accumulation of damaged proteins and or- lent of the apical epidermal ridge during limb development ganelles (Mizushima et al., 2008). Autophagy, as a method (Christensen and Tassava, 2000; Nye et al., 2003a). This of diet-induced modulations of homeostasis, is an evolution- structure delivers signals necessary for the formation and arily conserved mechanism using a highly conserved the maintenance of the blastema (Thornton, 1957; Nye et al., genetic circuitry. Besides maintaining metabolism, auto- 2003b). In Hydra very little is known about the role played by phagy can also lead to cell death, a mechanism that is the stretched ectodermal cells that rapidly cover the wound widely used across evolution in morphogenetic processes but pharmacological and RNAi experiments proved that (Melendez and Neufeld, 2008). In Hydra where the molecu- head regeneration does not proceed when wound healing lar components of the autophagy machinery are highly is de，cient (S. Chera, unpublished). In planarians the wound conserved, these studies indeed show that two distinct epidermis ful，lls a signaling function and a few candidate forms of autophagy can be activated, one physiological, genes were identi，ed in a systematic RNAi screen (Reddien observed during starvation (Buzgariu et al., 2008; Chera et al., 2005a). Hence, it might be possible to trace back some et al., 2009a) and a second, pathological one, when some conserved properties of the wound epidermis, possibly lost regulatory components are de，cient (Chera et al., 2006; or de，cient in species unable to repair or to regenerate. Galliot, 2006). Blastema Formation, an Adult Developmental TISSUE PLASTICITY IN REGENERATION Process
One striking aspect of regeneration is its evolutionary
Morphallaxis versus epimorphosis, a sterile debate distribution in the animal kingdom: the ability to anatomically
and functionally restore the lost body parts is widely, but Even though the outcome of regeneration is similar between nonuniformly spread in the animal kingdom (Fig. 2); also the species, that is, the de novo replacement of the organ or the ef，ciency and the regenerative strategies used vary not only missing body part, the mechanisms deployed for accom- among different phyla, but also between species of a given plishing this can be quite different among species and it was phylum (Sanchez Alvarado and Tsonis, 2006). The current so far impossible to outline a unifying view of the cellular and consensus view is that regeneration was quite common in molecular regeneration traits. However, the comparative early animal evolution but have undergone repeated loss analysis of regenerative contexts showed that the ability of or variations during evolution (Sanchez Alvarado, 2000; an organism to regenerate depends on its capacity to access Brockes and Kumar, 2008), possibly reflecting ecological a source of stem cells and/or to reprogram differentiated constraints (Bely and Nyberg, 2010). If true, then it is of cells (Brockes and Kumar, 2002; Odelberg, 2005; Poss, utmost interest to decipher the common themes, that is, the 2007; Birnbaum et al., 2008). These cells then adopt a core cellular and molecular processes underlying regenera- regeneration-speci，c behavior that was heavily investigated tion in vertebrates as well as in invertebrates (Galliot et al., in amphibians, ，sh, insects, and planarians. We will briefly 2008). We will discuss here several aspects of cellular review the general concepts that emerged from these plasticity that can impact regeneration but we will leave out studies.
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The regenerative strategies that lead to the rebuilding of body column, cells are already committed to a given pathway complex, multi-tissue structures are classically considered or on the way to differentiate (Steele, 2002). Thus, the as either morphallactic, that is, proceeding through re-pat- cellular contexts at the time of injury dramatically in！uence
terning the pre-existing tissue in the absence of cell prolifer- the regenerative route taken immediately after injury (Fig. 5). ation, or epimorphic, that is, relying on the proliferation of
undifferentiated progenitors that form a regeneration-spe- How to induce and grow a blastema? Two distinct ci，c structure, named the blastema (Morgan, 1901). This strategies to form a blastema were identi，ed: a direct one transient mass of mesenchymal proliferating cells is a self- relying on the recruitment of residing stem cells, and another organizing structure, which upon transplantation keeps the more indirect one, relying on cell plasticity, that is, on the memory of its origin and drives patterning, differentiation, dedifferentiation of adult cells located in the vicinity of the and morphogenesis of the regenerated structure (Stocum, wound (Fig. 5). Planarians illustrate the ，rst case where the 1968a,b). As a general rule, the blastema, which requires the formation of blastema requires the recruitment of ASCs AEC and some neurotrophic factors for its growth (Tassava named neoblasts (Reddien and Sanchez Alvarado, 2004). and Garling, 1979; Kumar et al., 2007), senses the disconti- Similarly, Xenopus tadpoles that regenerate their tail do not nuity with the remaining structure named stump. This sens- use dedifferentiation but rather recruit satellite cells, which ing promotes the establishment of the proximal and distal are small Pax7þ stem cells located in the basement boundaries and their confrontation leads to the regeneration membrane of the skeletal muscle (Slack et al., 2008). of intermediate structures that intercalate until the disconti- Recently, these satellite cells were also proposed to partici- nuity is ，lled (Nye et al., 2003b). These principles that were pate in blastema formation in amputated salamander limbs uncovered thanks to transplantation experiments of amphib- (Morrison et al., 2006). However, a large number of studies ian limb blastemas (Iten and Bryant, 1975; Stocum, 1975), indicate that blastema formation in urodeles regenerating were also identi，ed in insects that regenerate their appen- their appendages is predominantly indirect, relying on the dages (French et al., 1976; Nakamura et al., 2008), suggest- dedifferentiation of numerous cell types (multinucleated ing that the mechanisms were already at work in the last myocytes, ，broblasts chondrocytes, Schwann cells) into common bilateral ancestor. cycling progenitors (Hay, 1959; Geraudie and Singer, However, these two regenerative strategies, morphallac- 1981; Muneoka et al., 1986; Lo et al., 1993). The pluripo- tic versus epimorphic, might well be two extreme poles of tency of these progenitors was often assumed but never a continuum that would better represent the variable com- proven (for reviews see Brockes and Kumar, 2002; Bryant plexity of the multiple distinct regenerative contexts. For et al., 2002; Echeverri and Tanaka, 2002). example, if the nerve supply is de，cient, that is, the blastema Recently, the Tanaka’s group performed systematic cell does not grow properly, but in the presence of the AEC, a lineage tracing studies in transgenic animals, and showed miniature limb can regenerate, reminiscent of a morphallac- that the plasticity of these progenitors is actually more tic process (Nye et al., 2003b). Also in planarians, blastema restricted than anticipated: they keep the memory of their formation results from a mixed morphallactic-epimorphic cellular origin and re-differentiate in the regenerated struc- process that varies according to the site of the amputation ture according to this origin (Kragl et al., 2009). These direct (Salo and Baguna, 1984; Agata et al., 2007). Therefore, and indirect mechanisms of blastema formation are not opposing morphallaxis and epimorphosis actually promotes mutually exclusive and can be combined in different propor- a rather reductionist view of regeneration, which prevents its tions in distinct tissues of the same regenerative animal understanding (Agata et al., 2007). (Fig. 5). Also the dominant process might not be exclusive: it Following this vein, the question of the role of stem cells was recently proposed that neoblasts surviving nonlethal and proliferating cells in Hydra regeneration was recently irradiation might result from the dedifferentiation of radio- revisited. Classically Hydra regeneration is considered as resistant differentiated cells (Salvetti et al., 2009). If con- morphallactic, with two types of arguments supporting this ，rmed, this suggests that cellular events that are rare and statement: ，rst the absence of epithelial cell proliferation in thus dif，cult to detect, might actually become transiently head regenerating halves, at least on the ，rst day following accessible in restricted regeneration conditions. In Hydra bisection (Holstein et al., 1991), and second the fact that evidences for cell dedifferentiation are lacking. animals exposed to anti-mitotic drugs still regenerate their head (see in Bosch, 2007). However, in wild-type Hydra Transdifferentiation, A Special Case of Injury- interstitial progenitors and interstitial stem cells rapidly di- Induced Plasticity vide in head regenerating stumps after mid-gastric bisection
Some regenerative processes rely on transdifferentiation but not after decapitation, instead forming a blastema-like
of differentiated cells (as de，ned above) rather than on structure that drives head regeneration (Chera et al., 2009b
recruitment of stem cells or progenitor cells for blastema and unpublished). These results indicate that regeneration
growth. In homeostatic adult tissues, transdifferentiation is a in Hydra is more plastic than anticipated, following distinct
rare event, but its frequency increases upon injury. One of routes when the amputation level varies: at mid-gastric
the best-de，ned examples of organ regeneration through position head regeneration displays some features of
transdifferentiation in vertebrates is the induction of lens epimorphic regeneration, whereas after decapitation it is
from the pigmented epithelium of the newt iris. This process, morphallactic. The cellular backgrounds are indeed quite
named Wolf，an lens regeneration, was identi，ed in newt, different at these two positions; at the mid-gastric position, a
large number of cells are stem cells whereas in the upper ，shes, and chick embryo by the group of Goro Eguchi who
844 Mol Reprod Dev 77:837–855 (2010)
CELL PLASTICITY IN HOMEOSTASIS AND REGENERATION
Figure 5. Scheme depicting the three modules of a regeneration process. To regenerate a given structure (appendages, body part, organs, tissue), a variety of cellular processes, collectively forming the ‘‘induction module,’’ can trigger the structure-speci，c developmental process. The cellular processes of the induction module arise as a response to injury, in some contexts via the wound epidermis; they can be combined or not, providing multiple routes to bridge injury to regeneration. The homeostatic context at the time of injury largely influences this routing.
could observe it in vivo and reproduce it in vitro (Eguchi and in the de novo formed head region (Siebert et al., 2008). Kodama, 1993). In vivo the cells of the dorsal iris (but not the Nevertheless, these data do not tell us whether transdiffer- ventral one) are competent, activating the Six3 transcription entiation is a by-product of injury or a major player in factor to regenerate the lens after lentectomy (Grogg et al., regenerating Hydra. For example, does a head properly 2006). More generally in vertebrates, spontaneous transdif- regenerate when transdifferentiation is inhibited? To ad- ferentiation appears in contexts involving organ regenera- dress such question, the combination of cell lineage tracing tion, such as lens, retina, liver, pancreas (Slack, 2007). in transgenic animals to RNAi loss of function assays should In Hydra transdifferentiation appears common, as exem- help evaluate the contribution of transdifferentiation to Hydra pli，ed by the ganglia neurons that undergo this process at regeneration. By contrast in the jelly，sh Podocoryne
the time they get displaced along the oral-aboral axis, a (a species closely related to Hydra) transdifferentiation phenotype conversion (Bode, 1992). This phenotype con- appears as a driving force for regeneration: striated muscle version was identi，ed thanks to nerve-speci，c epitopes cells can be induced to differentiate to smooth muscle expressed in subsets of ganglia neurons at the extremities cells as well as neurons by disrupting the interactions but not in the body column. Surprisingly animals totally between cells and the extracellular matrix (Schmid and depleted in neuronal progenitors after nitrogen mustard or Alder, 1984; Schmid and Reber-Muller, 1995). This induced hydroxyurea treatments, can re-express some of these transdifferentiation event requires cell proliferation and is markers in apical neurons after decapitation, thus most likely able to support regeneration of the feeding organ (the arising in differentiated neurons that were not expressing manubrium).
them before bisection (Koizumi and Bode, 1986; Yaross In planarians, transdifferentiation of terminally differenti- et al., 1986). However, this phenotype conversion does not ated cells is poorly documented but the plasticity of post- ful，ll the criteria of transdifferentiation as changes in cellular mitotic cells in the blastema was established: these cells that morphology were not identi，ed. are already fate-committed can modify their fate according A more striking example of transdifferentiation in Hydra is to the surrounding tissue (Newmark et al., 2008). More that of ganglia neurons of the body column that after surprisingly, cytophotometric and karyological analyses bisection become epidermal sensory nerve cells in the have shown that germ cells can be recruited into the blaste- regenerated structure, head or foot (Koizumi et al., 1988; ma to adopt a somatic cell fate after injury (Gremigni et al., Koizumi and Bode, 1991). Such conversions require the 1980a,b). These examples of cellular plasticity in planarians differentiation of a new anatomical structure, the cilium, correspond to transdetermination rather than transdifferen- which is missing in ganglia neurons. More recently, the tiation events.
speci，c expression of GFP in gland cells of transgenic Hydra In conclusion, tissue restoration relying on transdifferen- was used to trace a similar transdifferentiation event: during tiation represents a case where a form of cell plasticity that is head regeneration after decapitation, the zymogen gland rare and unusual in homeostatic conditions is triggered cells of the body column are converted to mucous gland cells and enhanced by the injury stimulus. As such it provides
Mol Reprod Dev 77:837–855 (2010) 845
Molecular Reproduction & Development ALLIOT AND GHILA G
interesting possibilities for regenerative medicine. However, Regeneration, A Continuum of Development? the molecular mechanisms underlying transdifferentiation-
driven regeneration are still poorly understood. Planarians What is an adult organism? For a long time, regenera- and Hydra certainly provide fully appropriate systems to tion was considered as a developmental process that takes investigate how and when transdifferentiation contributes place during adulthood but is still tightly bound to organo- to regeneration. genesis and to a lesser extent to embryogenesis. This view led to the hypothesis that regeneration results from the reactivation of larval/fetal (possibly embryonic) develop-
mental processes in adulthood. However, this strict de，ni- ORIGIN(s) OF REGENERATION tion certainly does not cover the regeneration ，eld: ，rstly Regeneration, The Other Face of Asexual regenerative processes also take place in nonadult organ- Development? isms at various periods of their life cycle, and secondly The fact that the regenerative abilities are strongest in adulthood, which is de，ned by the acquisition of sexual species that can propagate asexually, like budding in Hydra, maturity, is an ambiguous concept. In fact, the state of ，ssion in planarians, or annelids (Sanchez Alvarado, 2000; adulthood (what we propose to name adulthoodness) at Brockes and Kumar, 2008; Bely and Nyberg, 2010) suggest the time regeneration is initiated in an organism is highly that regeneration and asexual reproduction might share variable: it varies between species as some species some evolutionary history. One possible scenario would be show the persistence of juvenile traits in adulthood, and it that regeneration evolved from asexual reproductive varies between individuals of a given species as aging mechanisms, conferring some adaptative advantages that obviously affects the regenerative potential. Therefore, might have sustained its perpetuation across evolution. ‘‘adulthoodness’’ should also be considered as an important Considering this view, Candia–Carnevali proposed to con- parameter to compare the different regenerative contexts sider ‘‘regeneration as the necessary and complementary and understand the principles of regeneration, as more developmental process associated with asexual reproduc- adulthoodness likely means less developmental activity and tion, in analogy with embryogenesis as being the develop- vice-versa. To take into account these two parameters, life mental strategy complementary to sexual reproduction’’ cycle stage and adulthoodness, we sorted a series of re- (Candia-Carnevali, 2006). The reproductive and regenera- generative contexts according to the developmental/adult tive properties of different protozoans, which generate two status of the organism at the time of injury (Table 1). new complete individuals by ，ssion or splitting, favor this hypothesis of a common origin for asexual reproduction and Sorting out of the regenerative processes according regeneration. The main difference between asexual repro-
duction and regeneration in protozoans as well as in Hydra to developmental criteria Regeneration of type 1
appears to be the stimulus triggering these two events: a includes all ‘‘regulative processes’’ that take place in the
favorable environmental conditions such as abundance of embryonic period as the half of the Xenopus embryo that food in the ，rst case, a deleterious incident, such as injury in regenerates a complete embryo (Reversade and De Rober- the latter one. In annelids, similar gene expression patterns tis, 2005) or the chick embryo regenerating its neural tube recorded during both ，ssion and regeneration were inter- (Ferretti and Whalley, 2008). As discussed above (see preted as an evidence of a shared genetic circuitry (Bely and Developmental plasticity), these cannot be considered sen- Wray, 2001). su stricto as regenerative processes since the developmen- However, several arguments challenge this proposal. tal program is broadly active at the time the structure is
amputated and re-built. Regeneration of type 2 corresponds First regeneration also takes place in numerous species
that do not display asexual reproduction, such as urodeles to ‘‘fetal/larval regeneration,’’ it occurs during organogenesis and teleost ，sh. But of course each of these two processes or larval stages but cannot occur after metamorphosis or likely had its own evolutionary history and the loss of asexual birth. Typical examples are insect larvae (the cricket nymph, reproduction across evolution might have occurred multiple the Drosophila) that regenerate their appendages (McClure times without affecting regenerative processes (Bely and and Schubiger, 2007; Nakamura et al., 2008) or the Xenopus Nyberg, 2010). Second although in Hydra the developmen- tadpole that regenerates its tail (Slack et al., 2008). After
metamorphosis or birth, the aging dimension should be tal programs that takes place when the head forms, appear
highly similar during regeneration, budding, and sexual taken into consideration as in most species juvenile organ-
isms certainly show a stronger regenerative potential than development (Gauchat et al., 1998; Technau and Bode,
1999), distinct signaling pathways appear to regulate initia- the sexually mature or aged individuals from the same tion of budding and initiation of regeneration in Hydra (Fabila species. Therefore, we consider ‘‘juvenile regeneration’’ as
et al., 2002; S. Chera et al., unpublished). And indeed only a separate type (type 3), taking place in fully developed budding and not regeneration is inhibited in starved Hydra organisms before they reach sexual maturity. (Fig. 4). Therefore, we assume the early signaling that links Type 4 regeneration is named ‘‘paedomorphic’’ and likely
injury to the reactivation of head formation might be shares similarities with types 2 and 3 as it takes place in
sexually mature organisms that are characterized by the regeneration-speci，c. According to that scenario, regener-
ation and asexual reproduction would converge on the same persistence of juvenile traits at adulthood. In such species, developmental program, here to form a new head, but would the developmental timing is shifted when compared to differ by the module that activates it. closely related species: either the sexual development takes
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