brigitte gallot and luiza ghila- cell plasticity in homeostasis and regeneration

8/3/2019 Brigitte Gallot and Luiza Ghila- Cell Plasticity in Homeostasis and Regeneration

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aged differentiated cells. This ongoing physiologicalreplacement process is named cell turnover. The adult stemcells (ASCs) play a key role in this turnover, although limitedto the organ or the tissue where they reside (Wagers andWeissman, 2004; Ohlstein and Spradling, 2006; Blanpainet al., 2007). As a classical scenario, ASCs divide through

asymmetric division, with one of the daughter cells keepingthe ‘‘stemness’’ status (self-renewal) whereas the secondone, no longera stem cell, undergoesa seriesof cell division,providing a transient amplifying stock that will subsequentlycommit to one or a series of differentiated fates (Raff, 2003).As a consequence three competitive processes regulatehomeostasis: cell death, cell proliferation, and cell differen-tiation. The study of their crosstalk in Drosophila imaginaldiscs showed how a coordinated cell–cell signaling tightlyregulates this competition in a given tissue (Moreno andBasler, 2004). In mammalian tissues, cell turnover occurs inepidermis, intestine, lung, blood, bone marrow, thymus,testis, uterus, and mammary gland with large variations inthe rate of cell turnover, from few days for the intestinalepithelium up to several months for the lung epithelium(Blanpain et al., 2007). In other organs (brain, heart, pan-creas, kidney, cornea, etc.), the physiological cell turnover islikely limited and/or very slow, making difficult the in vivomonitoring of the respective behaviors of stem cells anddying cells.

Similar to cell turnover, tissue repair also allows tissuereplacement but requires the damage-induced activation of programs that monitor cell proliferation and cell differentia-tion. Finally, regeneration of anatomical structures likeappendages, represent an even more complex process withformation of a transient proliferative structure, the blastema,and activation of a developmental program that leads to

restoration of original shape and function (Brockes andKumar, 2005). Both tissue repair and regeneration thataffect different tissue types and require cell replacement ona large-scale, are triggered by nonspecific and usuallyexogenous damage, whereas cell turnover is a process thatis endogenously initiated and restricted to a fraction of cells(Pellettieri and Sanchez Alvarado, 2007).

Nevertheless one can intuitively perceive a progressionfrom basic tissue self-renewal to tissue repair, reached bysome but not all organs, to regeneration, accessed by a‘‘happy few’’ elite of organs or structures. This view suggestsa possible continuum between the processes that regulateeach step, even though their complexity is supposed togradually increase. To challenge the solidity of this view,we review some results recently obtained in the paradig-matic Hydra model system. But before considering thedifferent forms of plasticity deployed in Hydra, we will firstdiscuss the origin and the current meaning of the concept of plasticity. Indeed, this concept is widely used by biologistsfrom different fields, but sometimes covering quite distinctmeanings.

The Ambiguities of the Concept of ‘‘Plasticity’’

The word ‘‘plasticity’’ (from Latin plasticus or Greekplastikos, ability to mold) refersto the‘‘capacity of distortablebodies to change their shape under the action of an external

force and to maintain the change after this force has ceasedto act’’ (from Littr e French dictionary, translated by Will et al.,2008).At thefirst look, this definitionapparently applies quitewell to the regenerative process, however, the usage of theword plasticity in biology is much broader, focusing on theability of livingorganisms to adapt to constraints by changing

their organization at a specific level, for example, evolution-ary, developmental, phenotypic, synaptic, cellular, and mo-lecular. As a consequence, the word ‘‘plasticity’’ shouldnever be used alone but always be specified by the levelwhere it applies (Pomerantz and Blau, 2004). Somescientists even proposed to apply to the concept of plasticityin biological systems a more ‘‘engineer-oriented’’ usage,restricting it to the contexts where lasting structural reorga-nization, that is, modifications of the material structure of the system (interface, connectivity network, constitutiveelements), are indeed proven, leaving out of plasticity theeffects of variability, flexibility, systematic variations, andvicarious (substituted) processes as these effects rather result from ‘‘operational’’ than structural changes (Willet al., 2008). We selected here few examples to discussthis view, certainly more rigorous or at least less metaphoric(following the words of Will et al., 2008) but as we will see,difficult to apply in some contexts.

Evolutionary plasticity is certainly the best example of plasticity with structural changes leading to lasting changes.The combination of genomic, genetic, and developmentalapproaches over the past 20 years have definitively proventhat variations in the genomic organization of the Hox geneclusters obviously lead to genetic reprogramming duringdevelopment and to species-specific modifications of thebody plan (Duboule, 2007). Developmental plasticity thatwas identified first in sea urchin embryos by Driesch in 1892,

and later in vertebrate embryos, refers to the embryonic potential for regulation as the embryoniccells at early stageshave the ability to change their fate to compensate for cellloss (Driesch, 1900). This potential, which accounts for theoccurrence of homozygous twins, is transient but can still beobserved at later stages in more specialized tissues as limbbuds (Summerbell, 1981) or neural crest cells (Vaglia andHall, 1999). Developmental plasticity, more recently namedtransfating (Keleher and Stent,1990), requires the activationof the gene regulatory network (GRN) that corresponds tothe new cell fate. Interestingly, in sea urchin embryo thisactivation apparently depends on inputs that are distinctduring normal and regulative developments (Ettensohnet al., 2007). If confirmed as a general rule, this would meanthat context-specific signals sensed at the ‘‘interface’’ of thesystem induce long-lasting structural reorganizations of thedeveloping organism.

Phenotypic plasticity is ‘‘the property of a given genotypeto produce different phenotypes in response to distinctenvironmental conditions’’ (Metcalf, 1906), with the firststudy of adaptive phenotypic plasticity described in thecrustacean Daphnia. However, the different phenotypesmight reveal an intrinsic ‘‘repertoire of competences’’ thatneed no structural changes to be expressed (Will et al.,2008). In the same year, 1906, the term neuroplasticity wasproposed by Ernesto Lugaro, a psychiatrist, who referred tothe changes in neural activity during psychic maturation,

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Molecular Reproduction & Development GALLIOT AND GHILA



learning processes, or post-damage recovery (Berlucchi,2002). During the first half of the 1900s, the concept of brainplasticity was rejected by the scientific community, as it wasunanimously accepted that the fully developed brainreached stability at adulthood, each region of the brainperforming specific function(s) that could not be modified.

In the 1960s, this view started to be challenged by experi-ments proving activity-dependent brain plasticity (Bennettet al., 1964; Bach-y-Rita et al., 1969). Synaptic plasticity, thecapability for a neuron to modify on the long term itselectrophysiological activity according to the stimuli it hadreceived,was first studied in themollusk Aplysia (Bruner andTauc, 1965; Kandel and Tauc, 1965). The choice of thismodel system was instrumental to establish the importanceof plasticity in the learning and memory processes as per-sistent modificationsof theactivity of the genetic circuitry arerequired to sustain changes in neurophysiological activity(Barco et al., 2006).

Cellular plasticity is directly related to the questionsaddressed in this review, that is, what conditions of tissuehomeostasis support a regenerative response. For thisreason, we will discuss here only the cellular plasticity of somatic cells (Fig. 1). As a first but rather rare strategydifferentiated cells can re-enter the cell cycle after injury,as exemplified by hepatocytes in mammals (Rabes et al.,1976). More frequently adult differentiated cells actuallydedifferentiate upon injury before entering an active cyclingphase to form a blastema (see below). But cells can alsoundergo metaplasia, that is,phenotypicallyconvert from onecell or tissue type into another, a process well known bypathologists, which actually covers a variety of processes.

Among those, transdifferentiation is defined by the factthat stably differentiated cells irreversibly change their fate,

that is, reprogram by acquiring a novel differentiated statuswith a specific molecular signature (Okada, 1991; Eguchiand Kodama, 1993). During that process, the cells may or may not traverse the cell cycle. Similarly cell fusion that, astransdifferentiation is also increased upon injury, might leadto reprogramming when twodistinctcell types fuse (Chiu andBlau, 1984; Pomerantz and Blau, 2004). Obtaining theexperimental proofs of transdifferentiation is often difficult,but at least morphological and molecular criteria as well ascell lineage relationships should clearly characterize the two

cell states before and after transdifferentiation (Wagers andWeissman, 2004; Slack, 2007). In fact, the most compellingevidence is provided by the transient co-expression of markers of the two differentiated cell states (Schmid andAlder, 1984).

More recently, it was possible to induce transdifferentia-

tion by overexpressing one or several cell-specific transcrip-tion factors that suffice to convert one cell type to another (Slack, 2007; Eberhard and Tosh, 2008; Zhou et al., 2008).Indeed nuclear reprogramming plays an essential role incellular plasticity and developmental biologists actuallyprovided the first experimental evidence of this event: theyshowed that nuclei isolated from mature somatic cellsand transplanted into enucleated Xenopus oocytes, couldreprogram and orchestrate the development of a frog(Gurdon et al., 1958). This surprising finding meant thatnuclei of terminally differentiated cells can become totipo-tent. Forty years later, the cloning of the sheep Dolly, alsoobtained by nuclear transfer from an adult somatic tissue,the mammary gland, confirmed this major finding inmammals (Wilmut et al., 1997). Actually, even nuclei frompost-mitotic neurons can be reprogrammed to drive thecomplete development of mice (Eggan et al., 2004).

Finally, since 2006 reprogramming of mature somaticcells can be pushed to the point where adult differentiatedcells directly reach an embryonic-like stemness thanks tothe co-expression of defined transcription factors withoutusing oocytes. Such cells, named induced pluripotent stemcells (iPSC), were obtained so far from fibroblasts(Takahashi and Yamanaka, 2006; Takahashi et al.,2007), lymphocytes (Hanna et al., 2008), keratinocytes(Aasen et al., 2008), cord blood cells (Haase et al., 2009),smooth muscle cells (Lee et al., 2010). Whatever the proce-

dure, reprogramming relies on epigenetic changes (Lohet al., 2008; Hochedlinger and Plath, 2009), which certainlycorrespond to ‘‘material’’ changes although not necessarily‘‘structural’’ changes.

Two Emerging Model Systems for InvestigatingHomeostasis and Regeneration

Two historical invertebrate model systems, Hydra andplanarians, were long recognized to be suitable for investi-gating the mechanisms supporting tissue homeostasis, ac-tive maintenance of patterning in adulthood as well ascomplex cellular reorganization to regenerate after injury.The freshwater polyp Hydra belongs to Cnidaria, a sister phylum to bilaterians, andthe flatworm planaria that belongsto Lophotrochozoa (see their respective phylogenetic posi-tions in Fig. 2) actually share five cellular and developmentalfeatures:

(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 stemcells, the endodermal epithelial stem cells, and theinterstitial stem cells).

(2) A stock of adult pluripotent stem cells that produce germcells and somatic cells throughout the life of the animals

Figure1. Thedifferent forms of cellular plasticitythat canbe observedor induced in differentiated somatic cells.

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CELL PLASTICITY IN HOMEOSTASIS AND REGENERATION



(the interstitial stem cells in Hydra; the neoblasts inplanarians), a situation quite unique among most animalphyla where germ cells usually segregate during earlyembryonic development.

(3) An efficient asexual reproduction mechanism, throughbudding in Hydra and fission in planaria.

(4) The amazing property to regenerate almost any missingpart of the body after injury.

(5) An apparent lack of aging, at least when the animals donot enter the sexual cycle (Martinez, 1998; Yoshidaet al., 2006; Pearson and Sanchez Alvarado, 2008).

However, Hydra and planarians are not genetically trac-table. The recent development of genomic, molecular andcellular tools promoted their emergence as modern modelsystems where the mechanisms of homeostasis and regen-eration can now be investigated thanks to RNA interference(RNAi) gene knocked down and transgenesis (see in Red-dien and Sanchez Alvarado, 2004; Galliot et al., 2006;Bosch, 2007; Bottger and Alexandrova, 2007; Salo et al.,2009). Homeostasis in planarians was recently reviewedin length (Pellettieri and Sanchez Alvarado, 2007;Handberg-Thorsager et al., 2008; Rossi et al., 2008) andwe will report here about the distinct forms of plasticitythat take place in adult Hydra polyps, first in response to

variations in the feeding diet, and second after bisection,when the animal survives the amputation stress and regen-erates the missing part. Given that most gene families thatcontrol cellular and developmental behaviors are presentand highly conserved in cnidarians (Putnam et al., 2007;Chapman et al., 2010), these forms of plasticity are likely notexotic and we will discuss the correspondences betweenthese changes and those observed in various bilaterianmodel systems.

TISSUE PLASTICITY IN HOMEOSTASIS

The Hydra body wall comprises two layers, ectodermaland endodermal, that together contain about a dozen celltypes. These cells derive from three distinct stem cellpopulations, ectodermal myoepithelial, endodermal myoe-pithelial, and interstitial cells (Dubel et al., 1987; Bode, 1996;Steele, 2002; Galliot et al., 2006). The spatial distribution of stem cells, progenitors, and differentiated cells along thepolyp occurs as a consequence of the continuous division of stem cells in the body column joined to the active migrationor the passive displacement of the committed/precursor cells in either apical or basal directions (Fig. 3A). Accordingto the cell types, the cells terminally differentiate during their

Figure 2. Wide distribution of the regenerative potentials across the animal kingdom. Phylogenetic tree of the animal species exhibiting a

regenerative potential after injury.





move or at their final position. Hence, the extremities, that is,the tentacles, the hypostome (dome apex surrounding themouth opening) andthe basal disk, aremade up of terminallydifferentiated cells that are continuously sloughed off. Thispermanent source of stem cells in the Hydra body columnlikely confers its unique cellular plasticity among multi-cellular adult organisms.

Budding and Autophagy, Two Ways to MaintainFitness in Hydra

In Hydra, the homeostasis mechanisms tightly link cellrenewal to an active maintenance of shape and fitness; thisis best illustrated by the adaptation of Hydra to feedingconditions, when the animal regulates its steady state bygrowing and budding with regular feeding. Upon starvation,

Figure 3. Cellular and morphological variations induced by the feeding diet in Hydra. A: Thehomeostasis of the Hydra adult polyp relies on the dynamic equilibrium between cell gain and cellloss. Cell proliferation takes place in the body column whereas cells differentiate when they migrate orget 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 offfrom theextremities and are replaced by the continuous influx of younger cells. B: The number of cells ina polyp is directly influenced bythe feedingdiet. Here, the cell numberby Hydra was plotted against thenumberof artemia given in thedailyfeeding (data taken from Bode et al., 1977).For each feeding perioda two-degree polynomial function was calculated to show the tendency of cell number changes.C: Morphogenesis in Hydra is directly influenced by the feeding diet. Upon starvation the animals stopbudding and rapidly activate autophagy to survive, reducing their size but keeping intact theirmorphology (red left panel). In steady-state condition,the animalsize is stableand asexualreproduction,

that is, budding, takes place with newbuds forming every 2 or 3 days (middle blue panel). In overfeedingconditions (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.





the steady state is no longer maintained and the animalsatrophy (Shostak, 1974). Indeed in the laboratory where it isrelatively easy to control the diet of a given population asHydra are fed with freshly hatched Artemia nauplii , Hydrareduces its size but maintains its shape, fitness, 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 themorphogenetic processes, namely the budding rate and themaintenance of patterning, as well as the cycling of theepithelial 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 tissuegrowth rate, and the cellular ‘‘surplus’’ is ‘‘eliminated’’through asexual reproduction—a fast process, whichcauses the growth of a bud on the parental body, whichitselfdoes notgrow.This budding process results in doublingthe animal number each 2–4 days; the distinct cell popula-tions composing the tissue mass indeed increase but their relative proportions remain stable (David and Campbell,1972; Bode et al., 1973).

By contrast in over-fed animals (over 25 artemia perday),both cell proliferation andbuddingare increased(Bodeet al.,1977; Otto and Campbell, 1977) and the tissue mass ex-ceeds the loss of tissuecaused by budding. Considering thatthe tissue loss at the base and at the tentacles is compara-tively low, the rate of budding predominantly regulates bothtissueloss and the length of the parent’s body. Some reportsactually suggest differences between the different Hydraspecies: the Japanese species H. magnipapillata elongatesits body column while the head remains at almost constant

size (Kroiher, 1999), whereas the European species H.vulgaris maintains its proportions (Muller, 1995). However,the steady state is never reached in overfed animals,which will eventually undergo heteromorphosis (bizarremorphological changes) and die (Bode et al., 1977).

Finally at low feeding level or under starvation conditions(0–1 artemia per day), Hydra polyps rapidly stop buddingand progressively decrease their size to about half with noalteration of their body shape or their fitness (Fig. 3C).Surprisingly, the relative sizes of the different cell popula-tions, as well as the total number of cells per animal, remainalmost constant (Bode et al., 1973). In fact, an imbalancebetween the decrease in polyp size and the cell cycle lengthwas observed, as cell proliferation initially remains roughlyconstant, leading to the overproduction of 10% cells per day(Bosch and David, 1984). This apparent contradiction wasexplained when Bosch and David found that apoptosis isactually rapidly induced upon starvation, in about 2 days(Fig. 4). As a consequence the supernumerary cellsproduced by cell proliferation during starvation becomeapoptotic and are engulfed by the neighboring epithelialcells, providing a regulatory mechanism for keeping moreor less stable the cell number (Bosch and David, 1984;Bottger and Alexandrova, 2007; Pauly et al., 2007).

However, the apoptotic process that affects less than 2%of the cells, remains stable over the starvation process(Bosch and David, 1984; Chera et al., 2009a). Therefore,

apoptosis is likely not sufficient to provide a long-standingenergy source. More recently, autophagy was detectedwhen feeding is stopped, first in the ectodermal, later on inthe endodermal myoepithelial cells (Buzgariu et al.,2008; Chera et al., 2009a). But in contrast to apoptosisthat remains constant during starvation, autophagy pro-gressively affects all epithelial cells, providing a source of nutrients over long periods of starvation (Fig. 4). Uponfeeding resumption, the animals immediately stop auto-phagy, start to re-grow, and recover their size and their ability to bud in several days. Thus, the data currentlyavailable suggest that Hydra adapts to low feeding dietthrough two distinct cellular mechanisms, autophagy for energy support and apoptosis for cell number.

Autophagy in Hydra Leads to Cell Death WhenDerepressed

A second form of autophagy was actually discovered bypure serendipidity when Kazal1, a Serine Protease INhibitor Kazal-type (SPINK ) gene, was silenced by repeatedly feed-ing Hydra with dsRNAs. Progressively an excessive auto-phagy was observed in the endodermal cells of these intactKazal1(RNAi) Hydra; large autophagosomes formed in thedigestive cells, progressively fusing and leading to cellshrinkage and cell death in several days (Chera et al.,

Figure 4. Cellular and developmental responses to starvation inHydra. Apoptosis is observed after 2 days of starvation, affecting lessthan2% of cells ofthe interstitial cell lineage even after a longperiodofstarvation. In contrast autophagy progressively affects all epithelialcells, reaching a plateau level after 11 days. These two cellularresponses immediately vanish when animals receive nutrients. Cellproliferation (not represented here) is not significantly affected uponstarvation (Bosch and David, 1984). At the developmental level,budding requires a regular feeding whereas regeneration does not.Indeed head regeneration is only slightly delayed in 17-days-starvedanimals when compared to daily fed animals (regeneration was nottested after periods of starvation longer than 17 days; Chera et al.,2009a).





2006). As previously mentioned homeostasis and buddingare tightly linked in Hydra and the first consequence of thisautophagy phenotype was to prevent budding and then toslowly induce animal death. These data indicate that thelevel of autophagy needs to be tightly controlled in steady-state homeostasis.

Interestingly, a deficit of Kazal-type protein activity alsoleads to excessive autophagy in mammals. In the Spink3 À/À

newborns, autophagosomes rapidly invades the exocrinepancreatic cells and the surrounding digestive cells(Ohmuraya et al., 2005). As in Hydra, these mice never gain weight and die in a couple of weeks. Therefore, in miceas in Hydra the Spink3 and Kazal1 proteins that areproduced by similar exocrine cells, that is, the zymogenpancreatic cells and the gland cells respectively, appear toplay a similar function, that is, to protect the cells thatproduce the digestive enzymes and the digestive cells fromself-digestion. This is the first example where a similar pathological cellular process regulated by related genefamilies can be traced from cnidarians to mammals.

Autophagy in eukaryotic cells involves the sequestrationand degradation of cytoplasmic organelles via the lysosomalpathway, as such it participates in the maintenance of cellular homeostasis by generating nutrients but also bypreventing the accumulation of damaged proteins and or-ganelles (Mizushima et al., 2008). Autophagy, as a methodof diet-induced modulations of homeostasis, is an evolution-arily conserved mechanism using a highly conservedgenetic circuitry. Besides maintaining metabolism, auto-phagy can also lead to cell death, a mechanism that iswidely used across evolution in morphogenetic processes(Melendez and Neufeld, 2008). In Hydra where the molecu-lar components of the autophagy machinery are highly

conserved, these studies indeed show that two distinctforms of autophagy can be activated, one physiological,observed during starvation (Buzgariu et al., 2008; Cheraet al., 2009a) and a second, pathological one, when someregulatory components are deficient (Chera et al., 2006;Galliot, 2006).

TISSUE PLASTICITY IN REGENERATION

One striking aspect of regeneration is its evolutionarydistribution in the animal kingdom: the ability to anatomicallyand functionally restore the lost body parts is widely, butnonuniformly spread in the animal kingdom (Fig. 2); also theefficiency and the regenerative strategies used vary not onlyamong different phyla, but also between species of a givenphylum (Sanchez Alvarado and Tsonis, 2006). The currentconsensus view is that regeneration was quite common inearly animal evolution but have undergone repeated lossor variations during evolution (Sanchez Alvarado, 2000;Brockes and Kumar, 2008), possibly reflecting ecologicalconstraints (Bely and Nyberg, 2010). If true, then it is of utmost interest to decipher the common themes, that is, thecore cellular and molecular processes underlying regenera-tion in vertebrates as well as in invertebrates (Galliot et al.,2008). We will discuss here several aspects of cellular plasticity that can impact regeneration but we will leave out

one major aspect of regeneration, which is the regulationand function of cell migration towards the wound.

Wound Healing in Regenerative andNonregenerative Contexts

The recovery of tissue integrity in response to environ-mental stress, injuries or diseases, encompassed by thenames tissue repair and regeneration, requires in multicel-lular organisms the activation of the wound healing process.Wound healing plays a decisive role in survival by rapidlycovering thewoundwith an epithelial layer that secures bodyintegrity and avoids tissue loss and infections. However inmammals, aging dramatically affects wound healing: em-bryos and fetuses are able to heal rapidly, efficiently, andwithout scarring, whereas in adults wound healing is oftenimperfect as in the case of the skin where it is limited toscarring (Redd et al., 2004). Indeed scarring and woundhealing are not identical as that latter process requires theunsilencing of repair genes through epigenetic reprogram-ming in the wound epidermis (Shaw and Martin, 2009). Anadditional level of complexity was observed in vertebratesthat regenerate appendages as the wound epidermis thatcovers the amputation plane thickens to form a structurenamed the apical epithelial cap (AEC), which is an equiva-lent of the apical epidermal ridge during limb development(Christensen and Tassava, 2000; Nye et al., 2003a). Thisstructure delivers signals necessary for the formation andthemaintenanceof theblastema(Thornton, 1957; Nyeet al.,2003b). In Hydra very little is known about the role played bythe stretched ectodermal cells that rapidly cover the woundbut pharmacological and RNAi experiments proved thathead regeneration does not proceed when wound healing

is deficient (S. Chera, unpublished). In planarians the woundepidermis fulfills a signaling function and a few candidategenes were identified in a systematic RNAi screen (Reddienet al., 2005a). Hence, it might be possible to trace back someconserved properties of the wound epidermis, possibly lostor deficient in species unable to repair or to regenerate.

Blastema Formation, an Adult DevelopmentalProcess

Morphallaxis versus epimorphosis, a sterile debateEven though the outcome of regeneration is similar betweenspecies, that is, the de novo replacement of the organ or themissing body part, the mechanisms deployed for accom-plishing this can be quite different among species and it wasso far impossible to outline a unifying view of the cellular andmolecular regeneration traits. However, the comparativeanalysis of regenerative contexts showed that the ability of an organism to regenerate depends on itscapacityto accessa source of stem cells and/or to reprogram differentiatedcells (Brockes and Kumar, 2002; Odelberg, 2005; Poss,2007; Birnbaum et al., 2008). These cells then adopt aregeneration-specific behavior that was heavily investigatedin amphibians, fish, insects, and planarians. We will brieflyreview the general concepts that emerged from thesestudies.





The regenerative strategies that lead to the rebuilding of complex, multi-tissue structures are classically consideredas either morphallactic , that is, proceeding through re-pat-terning the pre-existing tissue in the absence of cell prolifer-ation, or epimorphic , that is, relying on the proliferation of undifferentiated progenitors that form a regeneration-spe-

cific structure, named the blastema (Morgan, 1901). Thistransient mass of mesenchymal proliferating cells is a self-organizing structure, which upon transplantation keeps thememory of its origin and drives patterning, differentiation,and morphogenesis of the regenerated structure (Stocum,1968a,b).As a general rule, theblastema, which requires theAEC and some neurotrophic factors for its growth (Tassavaand Garling, 1979; Kumar et al., 2007), senses the disconti-nuity with the remaining structure named stump. This sens-ing promotes the establishment of the proximal and distalboundaries and their confrontation leads to the regenerationof intermediate structures that intercalate until the disconti-nuity is filled (Nye et al., 2003b). These principles that wereuncovered thanks to transplantation experiments of amphib-ian limb blastemas (Iten and Bryant, 1975; Stocum, 1975),were also identified in insects that regenerate their appen-dages (French et al., 1976; Nakamura et al., 2008), suggest-ing that the mechanisms were already at work in the lastcommon bilateral ancestor.

However, these two regenerative strategies, morphallac-tic versus epimorphic, might well be two extreme poles of a continuum that would better represent the variable com-plexity of the multiple distinct regenerative contexts. For example, if the nerve supplyis deficient, that is, theblastemadoes not grow properly, but in the presence of the AEC, aminiature limb can regenerate, reminiscent of a morphallac-tic process (Nye et al., 2003b). Also in planarians, blastema

formation results from a mixed morphallactic-epimorphicprocess that varies according to the site of the amputation(Salo and Baguna, 1984; Agata et al., 2007). Therefore,opposing morphallaxis and epimorphosis actually promotesa rather reductionist view of regeneration, which prevents itsunderstanding (Agata et al., 2007).

Following this vein, the question of the role of stem cellsand proliferating cells in Hydra regeneration was recentlyrevisited. Classically Hydra regeneration is considered asmorphallactic, with two types of arguments supporting thisstatement: first the absence of epithelial cell proliferation inhead regenerating halves, at least on the first day followingbisection (Holstein et al., 1991), and second the fact thatanimals exposed to anti-mitotic drugs still regenerate their head (see in Bosch, 2007). However, in wild-type Hydrainterstitial progenitors and interstitial stem cells rapidly di-vide in head regenerating stumps after mid-gastric bisectionbut not after decapitation, instead forming a blastema-likestructure that drives head regeneration (Chera et al., 2009band unpublished). These results indicate that regenerationin Hydra is more plastic than anticipated, following distinctroutes when the amputation level varies: at mid-gastricposition head regeneration displays some features of epimorphic regeneration, whereas after decapitation it ismorphallactic. The cellular backgrounds are indeed quitedifferent at these two positions; at the mid-gastric position, alarge number of cells are stem cells whereas in the upper

body column, cells arealready committedto a given pathwayor on the way to differentiate (Steele, 2002). Thus, thecellular contexts at the time of injury dramatically influencetheregenerative route taken immediately after injury(Fig. 5).

How to induce and grow a blastema? Two distinct

strategies to form a blastema were identified: a direct onerelying on the recruitment of residing stem cells, andanother more indirect one, relying on cell plasticity , that is, on thededifferentiation of adult cells located in the vicinity of thewound (Fig. 5). Planarians illustrate the first case where theformation of blastema requires the recruitment of ASCsnamed neoblasts (Reddien and Sanchez Alvarado, 2004).Similarly, Xenopus tadpoles that regenerate their tail do notuse dedifferentiation but rather recruit satellite cells, whichare small Pax7þ stem cells located in the basementmembrane of the skeletal muscle (Slack et al., 2008).Recently, these satellite cells were also proposed to partici-pate in blastema formation in amputated salamander limbs

(Morrison et al., 2006). However, a large number of studiesindicate that blastema formation in urodeles regeneratingtheir appendages is predominantly indirect, relying on thededifferentiation of numerous cell types (multinucleatedmyocytes, fibroblasts chondrocytes, Schwann cells) intocycling progenitors (Hay, 1959; Geraudie and Singer,1981; Muneoka et al., 1986; Lo et al., 1993). The pluripo-tency of these progenitors was often assumed but never proven (for reviews see Brockes and Kumar, 2002; Bryantet al., 2002; Echeverri and Tanaka, 2002).

Recently, the Tanaka’s group performed systematic celllineage tracing studies in transgenic animals, and showedthat the plasticity of these progenitors is actually morerestricted than anticipated: they keep the memory of their

cellular origin and re-differentiate in the regenerated struc-ture according to this origin (Kragl et al., 2009). These directand indirect mechanisms of blastema formation are notmutually exclusive and can be combined in different propor-tions in distinct tissues of the same regenerative animal(Fig. 5). Also the dominant process might not be exclusive: itwas recently proposed that neoblasts surviving nonlethalirradiation might result from the dedifferentiation of radio-resistant differentiated cells (Salvetti et al., 2009). If con-firmed, this suggests that cellular events that are rare andthus difficult to detect, might actually become transientlyaccessible in restricted regeneration conditions. In Hydraevidences for cell dedifferentiation are lacking.

Transdifferentiation, A Special Case of Injury-Induced Plasticity

Some regenerative processes rely on transdifferentiationof differentiated cells (as defined above) rather than onrecruitment of stem cells or progenitor cells for blastemagrowth. In homeostatic adult tissues, transdifferentiation is arare event, but its frequency increases upon injury. One of the best-defined examples of organ regeneration throughtransdifferentiation in vertebrates is the induction of lensfrom the pigmented epithelium of the newt iris. This process,named Wolffian lens regeneration, was identified in newt,fishes, and chick embryo by the group of Goro Eguchi who





could observe it in vivo and reproduce it in vitro (Eguchi andKodama, 1993). In vivo the cells of the dorsal iris (but not theventral one) are competent, activating the Six3 transcriptionfactor to regenerate the lens after lentectomy (Grogg et al.,2006). More generally in vertebrates, spontaneous transdif-ferentiation appears in contexts involving organ regenera-

tion, such as lens, retina, liver, pancreas (Slack, 2007).In Hydra transdifferentiation appears common, as exem-

plified by the ganglia neurons that undergo this process atthe time they get displaced along the oral-aboral axis, aphenotype conversion (Bode, 1992). This phenotype con-version was identified thanks to nerve-specific epitopesexpressed in subsets of ganglia neurons at the extremitiesbut not in the body column. Surprisingly animals totallydepleted in neuronal progenitors after nitrogen mustard or hydroxyurea treatments, can re-express some of thesemarkers in apicalneurons after decapitation, thus most likelyarising in differentiated neurons that were not expressingthem before bisection (Koizumi and Bode, 1986; Yarosset al., 1986). However, this phenotype conversion does notfulfill the criteria of transdifferentiation as changes in cellular morphology were not identified.

A more striking example of transdifferentiation in Hydra isthat of ganglia neurons of the body column that after bisection become epidermal sensory nerve cells in theregenerated structure, head or foot (Koizumi et al., 1988;Koizumi and Bode, 1991). Such conversions require thedifferentiation of a new anatomical structure, the cilium,which is missing in ganglia neurons. More recently, thespecific expression of GFP in gland cells of transgenic Hydrawas used to trace a similar transdifferentiation event: duringhead regeneration after decapitation, the zymogen glandcells of thebody columnare convertedto mucousglandcells

in the de novo formed head region (Siebert et al., 2008).Nevertheless, these data do not tell us whether transdiffer-entiation is a by-product of injury or a major player inregenerating Hydra. For example, does a head properlyregenerate when transdifferentiation is inhibited? To ad-dress such question, the combination of cell lineage tracing

in transgenic animals to RNAi loss of function assays shouldhelp evaluate the contribution of transdifferentiation to Hydraregeneration. By contrast in the jellyfish Podocoryne(a species closely related to Hydra) transdifferentiationappears as a driving force for regeneration: striated musclecells can be induced to differentiate to smooth musclecells as well as neurons by disrupting the interactionsbetween cells and the extracellular matrix (Schmid andAlder, 1984; Schmid and Reber-Muller, 1995). This inducedtransdifferentiation event requires cell proliferation and isable to support regeneration of the feeding organ (themanubrium).

In planarians, transdifferentiation of terminally differenti-ated cells is poorly documented but the plasticity of post-mitotic cells in the blastema wasestablished: these cells thatare already fate-committed can modify their fate accordingto the surrounding tissue (Newmark et al., 2008). Moresurprisingly, cytophotometric and karyological analyseshave shown that germ cells can be recruited into the blaste-ma to adopt a somatic cell fate after injury (Gremigni et al.,1980a,b). These examples of cellular plasticity in planarianscorrespond to transdetermination rather than transdifferen-tiation events.

In conclusion, tissue restoration relying on transdifferen-tiation represents a case where a form of cell plasticity that israre and unusual in homeostatic conditions is triggeredand enhanced by the injury stimulus. As such it provides

Figure5. Schemedepicting thethreemodules of a regenerationprocess. To regenerate a given structure (appendages,body part, organs, tissue),a variety of cellular processes, collectively forming the ‘‘induction module,’’ can trigger the structure-specific developmentalprocess. Thecellularprocessesof theinduction modulearise as a response to injury, in some contexts viathe wound epidermis; they can be combined or not,providingmultiple routes to bridge injury to regeneration. The homeostatic context at the time of injury largely influences this routing.





interesting possibilities for regenerative medicine. However,the molecular mechanisms underlying transdifferentiation-driven regeneration are still poorly understood. Planariansand Hydra certainly provide fully appropriate systems toinvestigate how and when transdifferentiation contributesto regeneration.

ORIGIN(s) OF REGENERATION

Regeneration, The Other Face of AsexualDevelopment?

The fact that the regenerative abilities are strongest inspecies that can propagate asexually, like budding in Hydra,fission in planarians, or annelids (Sanchez Alvarado, 2000;Brockes and Kumar, 2008; Bely and Nyberg, 2010) suggestthat regeneration and asexual reproduction might sharesome evolutionary history. One possible scenario would bethat regeneration evolved from asexual reproductivemechanisms, conferring some adaptative advantages thatmight have sustained its perpetuation across evolution.Considering this view, Candia–Carnevali proposed to con-sider ‘‘regeneration as the necessary and complementarydevelopmental process associated with asexual reproduc-tion, in analogy with embryogenesis as being the develop-mental strategy complementary to sexual reproduction’’(Candia-Carnevali, 2006). The reproductive and regenera-tive properties of different protozoans, which generate twonew complete individuals by fission or splitting, favor thishypothesis of a common origin for asexual reproduction andregeneration. The main difference between asexual repro-duction and regeneration in protozoans as well as in Hydraappears to be the stimulus triggering these two events: a

favorable environmental conditions such as abundance of food in the first case, a deleterious incident, such as injury inthe latter one. In annelids, similar gene expression patternsrecorded during both fission and regeneration were inter-preted as an evidence of a shared genetic circuitry (Bely andWray, 2001).

However, several arguments challenge this proposal.First regeneration also takes place in numerous speciesthat do not display asexual reproduction, such as urodelesand teleost fish. But of course each of these two processeslikely hadits ownevolutionary history and theloss of asexualreproduction across evolution might have occurred multipletimes without affecting regenerative processes (Bely andNyberg, 2010). Second although in Hydra the developmen-tal programs that takes place when the head forms, appear highly similar during regeneration, budding, and sexualdevelopment (Gauchat et al., 1998; Technau and Bode,1999), distinct signaling pathways appear to regulate initia-tion of budding and initiation of regenerationin Hydra (Fabilaet al., 2002; S. Chera et al., unpublished). And indeed onlybudding and not regeneration is inhibited in starved Hydra(Fig. 4). Therefore, we assume the early signaling that linksinjury to the reactivation of head formation might beregeneration-specific. According to that scenario, regener-ation andasexual reproduction would converge on the samedevelopmental program, here to form a new head, but woulddiffer by the module that activates it.

Regeneration, A Continuum of Development?

What is an adult organism? For a long time, regenera-tion was considered as a developmental process that takesplace during adulthood but is still tightly bound to organo-genesis and to a lesser extent to embryogenesis. This viewled to the hypothesis that regeneration results from thereactivation of larval/fetal (possibly embryonic) develop-mental processes in adulthood. However, this strict defini-tion certainly does not cover the regeneration field: firstlyregenerative processes also take place in nonadult organ-isms at various periods of their life cycle, and secondlyadulthood, which is defined by the acquisition of sexualmaturity, is an ambiguous concept. In fact, the state of adulthood (what we propose to name adulthoodness) atthe time regeneration is initiated in an organism is highlyvariable: it varies between species as some speciesshow the persistence of juvenile traits in adulthood, and itvaries between individuals of a given species as aging

obviously affects the regenerative potential. Therefore,‘‘adulthoodness’’ should also be considered as an importantparameter to compare the different regenerative contextsand understand the principles of regeneration, as moreadulthoodness likely means less developmental activity andvice-versa. To take into account these two parameters, lifecycle stage and adulthoodness, we sorted a series of re-generative contexts according to the developmental/adultstatus of the organism at the time of injury (Table 1).

Sorting out of the regenerative processes accordingto developmental criteria Regeneration of type 1includes all ‘‘regulative processes’’ that take place in the

embryonic period as the half of the Xenopus embryo thatregenerates a complete embryo (Reversade and De Rober-tis, 2005) or the chick embryo regenerating its neural tube(Ferretti and Whalley, 2008). As discussed above (seeDevelopmental plasticity ), these cannot be considered sen-su stricto as regenerative processes since the developmen-tal program is broadly active at the time the structure isamputated and re-built. Regeneration of type 2 correspondsto ‘‘fetal/larval regeneration,’’ it occurs during organogenesisor larval stages but cannot occur after metamorphosis or birth. Typical examples are insect larvae (the cricket nymph,the Drosophila) that regenerate their appendages (McClureandSchubiger, 2007; Nakamura et al., 2008) or the Xenopustadpole that regenerates its tail (Slack et al., 2008). After metamorphosis or birth, the aging dimension should betaken into consideration as in most species juvenile organ-isms certainly show a stronger regenerative potential thanthe sexually mature or aged individuals from the samespecies. Therefore, we consider ‘‘juvenile regeneration’’ asa separate type (type 3), taking place in fully developedorganisms before they reach sexual maturity.

Type 4 regeneration is named ‘‘paedomorphic’’ and likelyshares similarities with types 2 and 3 as it takes place insexually mature organisms that are characterized by thepersistence of juvenile traits at adulthood. In such species,the developmental timing is shifted when compared toclosely related species: either the sexual development takes





place too early (progenesis) or somatic development isslowed down and not achieved at the time sexual maturityis established (neoteny). Consequently, these animalsreach sexual maturity without displaying the full panel of somatic ancestral features of adulthood (Gould, 2000).There are two good examples of paedomorphic specieswith high regenerative potential: the Hydra polyps that aresexually mature as polyps and not as medusa, which is theancestral adult status in medusozoans (Hydra actually lostthe medusa stage) and the neotenic Axolotl salamander thatdoes not undergo metamorphosis, remaining aquatic in-stead of terrestrial all along its life. The high regenerativepotential of Axolotl reflects an easier access to developmen-tal programs despite the sexually mature status; this poten-tial was actually proposed to be secondarily acquired

(Tanaka and Ferretti, 2009). Finally, a 5th type is ‘‘adultregeneration’’ as it takes place in animals that reached bothsexual and somatic maturity. In fact adult regeneration ishighly variable, either complete as in planarians, or quitebroad as in teleost fish and urodeles that regenerateappendages, nervous system, organs, or restricted to tissuerepair as in most mammals.

The developmental part of the regeneration pro-gram. The correlation between the strength of the regen-eration potential and the developmental status indeedindicates that when the developmental programs close, theregenerative potential is drastically altered in most species.Therefore the analysis of regeneration in developing organ-isms offers the possibility to understand how this property issilenced during development, at the time of metamorphosisor when sexual maturity is established. If the ‘‘developmentcontinuum’’ hypothesisholds true then the genetic circuitriessupporting development and regeneration should be highlysimilar if not identical. Indeed the regenerative re-patterningmechanisms often deploy developmental genes. Inurodeles (paedomorphic or adult regeneration) as in the

Xenopus tadpole (larval regeneration) the genetic programssupporting limb development and limb regeneration sharesimilarities, although restricted to the last phase of thedevelopmental and regenerative processes, at the time thelimb bud forms and grows (Bryant et al., 2002; Pearl et al.,

2008). Similar conclusion was obtained in Xenopus tadpolesafter induction of tail regeneration at the refractory period(Beck et al., 2003).

Hence, one of the questions currently under investigationis whether regeneration partially reiterates embryogene-sis and/or organogenesis, following generic-patterningmechanisms, or alternatively makes use of regeneration-specific circuitries that include developmental genes(Birnbaum and Sanchez Alvarado, 2008; Brockes andKumar, 2008). There is no clear answer to this question asin fact only few regeneration-specific mutants were charac-terized (Nechiporuk et al., 2003; Behra et al., 2009) andregeneration-specific circuitries remain to be identified.Obviously adult regeneration contexts (type 5) are mostfavorable to characterize such circuitries.

In contrast to the late phase of regeneration character-ized by morphogenesis and growth, the cellular and geneticprograms at work at the initial and intermediate phases of regeneration (wound healing and blastema formation,respectively) do not match the determination and initiationphases of embryogenesis (Brockes and Kumar, 2005). Thisresult is actually not so surprising as regeneration anddevelopment occur in distinct contexts andat distinct scales;during regeneration, in response to the tissue loss, the cellsreorganize within pre-existent tissues in injury-inducedstress conditions, whereas during development the cellsmigrate and differentiate in specific tissues, which formstructures following strict temporal-spatial rules. At the mo-lecular level, the injury response recruits the stressresponsepathways (Pearl et al., 2008), whereas blastema formationrelies on signals with no or limited activity during develop-ment (Kumar et al., 2007; Yin and Poss, 2008). As aconclusion, regeneration certainly does not simply recapit-ulate development even though it makes use of tools previ-ously used during embryonic and fetal/larval development.

Regeneration, A Continuum of Homeostasis?

The recent tremendous development of stem cell biologyand cell plasticity fields helped take a novel view point, nolonger considering regeneration as strictly developmental,but also as a cell biology problem where the dynamics

TABLE 1. Table Sorting the Different Types of RegenerationAccording to the Developmental Status of the Organismat the TimeRegeneration is Initiated. Only Types 4 and 5 Take Place in Sexually Mature Organisms.

Regeneration typesDevelopmental status

at the time regeneration occurs Representative species! regenerated structure

1. Regulative processes Embryogenesis Xenopus embryo! full embryoChick embryo!neural tube

2. Fetal/larval regeneration Organogenesis or larvaldevelopment (before metamorphosis)

Drosophila larva!appendages,Cricket nymph!appendages, Xenopus tadpole! tail

3. Juvenile regeneration Fully developed but sexually immature organisms Human infants! distal finger phalange4. Paedomorphic regeneration Adult organisms with larval/juvenile traits

as a result of progenesis or neotenyHydra polyps!bodyAxolotl! limbs, tail

5. Adult regeneration Fully developed and sexually mature organisms Planarians!bodyEchinoderms! intestine, armsZebrafish! fins, heartNewt! limbs, jaws, lensDeer ! antlers

Only types 4 and 5 take place in sexually mature organisms.





of tissues in steady-state conditions would dramaticallyinfluence the response of the organ or the body to stressand injury within a given time frame (Birnbaum and SanchezAlvarado, 2008). This clearly provides a different perspec-tive to decipher the enigma of the variability in the regener-ative potential. In the next sections, we will discuss how

homeostasis might impact on regeneration.

Are the mechanisms that maintain homeostasis re-cruited in response to injury? Autophagy in regenera-tion. The Hydra model systemis wellsuitedto testthe roleof processes that maintain homeostasis during regeneration.As reported above, autophagy is an essential process tosurvive long periods of starvation in Hydra (Buzgariu et al.,2008; Chera et al., 2009a). When Hydra are exposed for 2 hr at the time of bisection to pharmacological drugs that induceor inhibit autophagy, head regeneration is only slightly de-layed, suggesting that transient modulations in the level of autophagy do not dramatically affect regeneration. In con-trast in Kazal1 RNAi knocked-down Hydra the amputationstress appears to immediately enhance the pre-existinglevel of autophagy in the endodermal layer. Interestingly,this immediate post-injury autophagy is reversible in a fewhours if the pre-injury autophagy is moderate, but no longer reversible when the pre-injury autophagy is high. Therefore,high levels of autophagy in the endoderm at the time of bisection are not compatible with the stress of the amputa-tion (Chera et al., 2006; Galliot, 2006).

Consequently, the mechanisms that limit the level of autophagy after amputation certainly play an essentialcytoprotective function. Similar mechanisms apply in Hydraand mammals, one of them would be the up-regulation of protease inhibitors in the injured region (Neuschwander-

Tetri et al., 2004). These data suggest that two distinct typesof autophagy with opposite regulations develop in chronicand acute contexts, a slow and positive one that progres-sively increases during diet restriction to support tissuesurvival, a fast and negative one that rapidly leads to celldeath after injuryif notrepressed. It is currently notclear howmuch is shared between these two types of autophagy. Inplanarians, a report suggests some role for autophagyduring regeneration and starvation (Gonzalez-Estevezet al., 2007).

Apoptosis in regeneration. Apoptosis also seems to playquite different roles in homeostasis and regeneration inHydra. The wave of apoptosis that takes place in head-regenerating tips immediately after mid-gastric section,affects about 50% of the cells in the first hour following injuryand is critical to induce the proliferation of the surroundingprogenitor cells as apoptotic cells release signaling mole-cules as Wnt3 (Chera et al., 2009b). By contrast less than1% of the cells areapoptotic in homeostatic conditions (up to2% during starvation); these cells are distributed along thebody columnand are supposedto maintain the cell mass inasteady state (Bosch and David, 1984).

Apoptosis is actually emerging as an important processto bridge injury to regeneration: it was observed duringplanarian regeneration (Hwang et al., 2004; Pellettieriet al., 2009) and is required during the first day of tail

regeneration in the Xenopus tadpole (Tseng et al., 2007).In two other contexts, wing discs of Drosophila larvae andhead regenerating tips in Hydra, injury-induced apoptoticcells were actually shown to induce compensatory prolifer-ation by releasing signaling molecules as Wg/Wnt3, Dpp or Hedgehog (Huh et al., 2004; Perez-Garijo et al., 2004; Ryoo

et al., 2004; Fan and Bergmann, 2008; Chera et al., 2009b).Two non-mutually exclusive mechanisms were pro-

posed as to the role of the apoptotic cells during regenera-tion: eitherthe recruitment of stem cells andprogenitorsvia adirect signaling and/or the selective destruction of cells thatnormally exert a negative pressure on the cycling activity of progenitors and stem cells (Simon et al., 2009). A recentstudy that investigatedthe function of apoptotic cells in mice,showed that indeed injury-induced cell death efficientlytriggers cell proliferation in vitro and in vivo, as well as tissuerepair in skin and liver (Li et al., 2010). These authorsidentified the activated caspases 3 and 7 as key players togenerate arachidonic acid, itself converted into prostaglan-dins that stimulate proliferation of stem cells; they namedthis mode of signaling the ‘‘phoenix rising’’ pathway.As apoptosis by itself is an extremely fast process (about1 hr), its role waslikely overlooked in thepast butrecent toolssuch as apoptosis sensors should soon help identify theregulation(s) and action(s) of this pathway across variousregenerative contexts.

Adult stem cells as a direct support to build a regen-erative response. A determining factor to potentiatetissue repair and regenerationis the intensity of self-renewalin homeostasis. The direct influence of the ASCs on theregenerative response to injury or stress support the

‘‘homeostasis continuum’’ hypothesis and open new hopesfor establishing a regenerative medicine. Hydra and planar-ians provide robust experimentalmodel systems to decipher how the biology of ASCs impact on the regenerationpotential.

ASCs in the Hydra and planarian regenerative responses.Hydra regeneration requires complex and variable interac-tions between the epithelial and interstitial stem cells. As afruitful experimental approach, interstitial stem cells caneasily be eliminated either after a short antimitotic treatmentor after heat-shock in the sf-1 temperature-sensitive mutant,producing ‘‘epithelial’’ animals unable to catch their food butable to bud and regenerate although with less efficiency(Campbell, 1976; Marcum et al., 1980). This proves thatepithelial stem cellscan drivemorphogenesisin the absenceof interstitial stem cells. Indeedepithelial cells were shown toproduce signaling molecules and epitheliopeptides involvedin morphogenesis (Fujisawa, 2003; Guder et al., 2006;Lengfeld et al., 2009). However, the interstitial stem cellsalso likely play a role in this flexible scenario; they producesignaling peptides (Schaller et al., 1989), they interact withepithelial cells to finely tune their morphogenetic potential(Sugiyama and Wanek, 1993), they appear essential totrigger the head regeneration program after mid-gastricsection (Chera et al., 2009b), they participate in headhomeostasis and head regeneration by regulating apical





neurogenesis (Miljkovic-Licina et al., 2007). Transplantinginterstitial stem cells into epithelial Hydra demonstrated their multipotency and the plasticity of nerve precursors (Holsteinand David, 1990b; Fujisawa, 1992; Minobe et al., 1995).Similarly, experiments combined to BrdU labeling identifiedpopulations of interstitial cells cycling at distinct rates

(Holstein and David, 1990a). Therefore, future studiesshould tell us more about the plasticity of the epithelial andinterstitial stem cells and about the way they interact to formstem cell niches that contribute to regeneration in Hydra(Galliot et al., 2006; Wittlieb et al., 2006; Bosch, 2009).

In planarians the rapid proliferation of neoblasts isessential to mount the regenerative response; their role wasrecognized decades ago, thanks to irradiation experimentsthat eliminate them (Wolff and Dubois, 1948; Salo andBaguna, 1985) and more recently to BrdU-labeling experi-ments that proved that neoblasts are indeed the only cells todivide in planarians (Newmark and Sanchez Alvarado,2000). Neoblasts form a heterogeneous cell population(Reddien et al., 2005b; Hayashi et al., 2006) and useevolutionarily conserved genetic programs to regulate stem-ness (Reddien et al., 2005b; Guo et al., 2006; Rossi et al.,2007; Eisenhoffer et al., 2008). As an example, the PTEN/TOR pathway, a critical regulator of self-renewal of stemcells (Hill and Wu, 2009), is required for regeneration. InPTEN RNAi knocked-down planarians, a pseudo-metastaticprocess was observed with neoblast proliferation, disorga-nization of differentiated tissues andloss of basal membraneintegrity, indicating that the biology of stem cells is dramati-cally impaired (Oviedo et al., 2008). In Hydra the PTEN/TORpathway is present (Chera et al., 2009a), but its function wasnot tested yet. Further comparative analyses should tell usmore about the regulatory pathways shared between Hydra

stem cells and planarian neoblasts.

Adult neurogenesis from cnidarians to mammals. Onetissue that was thought for long to be refractory to tissuerepair is the central nervous system (CNS), which oncedeveloped would not be able to self-renew. However, adultneurogenesis was recently identified in mammals, includinghumans, in two regions of the CNS, the hypothalamus andthe olfactory bulb (Suh et al., 2009). The plasticity of theneural stem cells identified in these two locations mightprovide a source for regenerative medicine. In amphibians,reptiles, and teleost fish adult neurogenesis is even morewidely distributed (Kaslin et al., 2008; Zupanc, 2008), asso-ciated in some species to the regeneration of the CNS

(Tanaka and Ferretti, 2009). Also urochordates as Cionaexhibit a strong but age-dependent capacity for regeneratingtheir CNS (Dahlberg et al., 2009; Auger et al., 2010). Finallyin Hydra, all cell types of the adult nervous system (sensorycells, ganglia cells, mechano-receptor cells) are continu-ously replaced in homeostatic conditions and the nervoussystem that is denser and organized at the apical pole is fullyregenerated in few days after amputation (Bode, 1996;Galliot et al., 2009).

The molecular and cellular basis of this plasticity is notknown yet but some candidates were already identified. For example, the maintenance of adult apical neurogenesis andthe de novo neurogenesis during head regeneration are

dramatically impaired when the Gsx paraHox transcriptionfactor, which is expressedin neuronal progenitors andapicalneurons, is knocked-down by RNAi (Miljkovic-Licina et al.,2007). Interestingly, this transcription factor is also involvedin neurogenesis in developing mice,specifyingthe identity of a subset of telencephalic progenitors during development

(Yun et al., 2003).It would be of interest to know whether Gsx orthologs also play a role in vertebrate adult neurogenesis.Similarly, the CREB transcription factor appears as a keyregulator of neurogenesis from Hydra (Chera et al., 2007) tozebrafish and mice (Dworkin et al., 2007; Dworkin et al.,2009).

In addition some of these model systems such as thezebrafish allow for comparison of developmental and adultneurogenesis (Zupanc, 2008) and to characterize the stemcell niches where neuronal progenitors keep proliferating inadulthood (Kaslin et al., 2009). This is of utmost importanceas recent studies indeed revealed clear differences betweenembryonic and ASCs, likely reflecting the age-dependentvariations of the mechanisms supporting stem cell function(Levi and Morrison, 2008; Suh et al., 2009). In short self-renewal becomes deficient over time, impacting on the sizeof the stem cells stock and thus reducing the regenerationpotential. Hydra oligactis, a species where aging is observedwhen sexual differentiation is induced (Yoshida et al., 2006)provides a suitable experimental framework to test themechanisms that link aging process, adult neurogenesis,and regeneration of the nervous system.

A MODEL OF MODULAR ORGANIZATION FORREGENERATION

Reviewing the various aspects of plasticity, we haveinspected many pieces of the homeostasisand regenerationpuzzles. In this last section, we will try to see how thesepieces might work together to mount a regenerativeresponse, obeying some rules that might apply from Hydrato vertebrates.

The Early Phase of Regeneration Draws Its ToolsFrom Homeostatic Plasticity

The cellular properties of the immediate andearly phasesof regeneration that are distinct from embryogenesis or organogenesis by a number of criteria (Brockes and Kumar,2005), seem to share more with the dynamics of homeo-stasis. Typically only organisms, organs or tissues that

maintain a dynamic homeostasis based on cell renewal or cell plasticity, are able to launch a regenerative responseafter injury. However, this injury-dependent regenerativeresponse takes place neither at the same scale nor at thesame speed when compared to homeostasis: processesthat are extremely rare and difficult to observed in homeo-static conditions occur within a short period of time after injury, often affecting a much larger number of cells. For example, injury promotes the conditions for generating aregenerative response by dramatically enhancing the levelof apoptosis, by promoting transdifferentiation, by inducingdedifferentiation, or by pushing stem cells, progenitor cells,or even differentiated cells to cycle. These two criteria, the





speed and the magnitude of the injury-induced cellular processes, appearas a commonthemebetween thevariousregenerative contexts rather than the processes them-selves. Finally, some aspects of regeneration such as posi-tional memory, nerve dependence of the blastema growthappear neither as a continuum of homeostasis, nor as a

continuum of development, but fully specific to regeneration(Kumar et al., 2007). Whether they obey common rules invertebrate and invertebrate contexts is currently not known.

The Late Phase of Regeneration MimicsDevelopment

As discussed above, the correlation between asexualreproduction and regeneration in a number of species(cnidarians, planarians, annelids) might reflect a commonorigin for these two processes. This question can be ad-dressed in Hydra where head formation can easily becompared in three distinct adult developmental contexts,budding, head regeneration after decapitation, head regen-eration after mid-gastric section. Preliminary results indicatethat asexual reproduction and head regeneration follow asimilar structure, formed of successive modules. The lastmodule that we name ‘‘development-like module,’’ supportsthe formation of the new head and appears highly similar if not identical between asexual reproduction and the twotypes of regeneration. Moreover, these adult developmentalprograms seem to closely resemble the embryonic/fetalprograms involved in head formation (Technau and Bode,1999). Therefore, as in vertebrates, the late phase of regen-eration, that is, the differentiation of the de novo structureappears to re-apply with similar rules, the tools previouslyused during development. If confirmed, this conservation

indicates that the development-like module of a specificstructure remained highly constrained across evolution.

Regeneration Results From the SequentialActivation of Homeostasis-Derived andDevelopment-Like Modules

By contrast the mechanisms that lead to the activation of the developmental module seems to be multiple. As they allprecede the differentiation of the de novo structure, we havegrouped them into the ‘‘induction module.’’ In Hydra theinduction module corresponds to the induction of budding,that is, the formation of the bud spot on the parental polyp, or the activation of the regeneration program through the injuryresponses (Fig. 5). As we saw, these injury responses areactually quite different after decapitation or after mid-gastricbisection (morphallactic or epimorphic-like, respectively).Therefore, we suspect that the variability of the inductionmodule directly reflects the parameters that define eachcomponent of the homeostatic plasticity within a giventissue, a given organ (transdifferentiation, dedifferentiation,recruitment of stem cells, proliferation of differentiatedcells, etc.). These components constrain the regenerativeplasticity that will be developed upon injury.

Finally, the immediate module is the wound healingresponse (of course absent when injury or stress are lackingas during budding). Its major regenerative function, besides

preserving tissue, organ, structure integrity, would be torapidly amplify the components of homeostatic plasticity toconvert them to regenerative plasticity as defined by theinduction module. Whether the impact of wound healing onthe activation of the induction module is fixed or plastic isunknown. This modular organization of the regenerative

process resulting from a combination of highly constraineddevelopment-like modules that are structure-specific andmuch more variable homeostatic-dependent inductionmodules can account for the diversity of the regenerativeresponses, sometimes even in the same organism asobserved in Hydra regenerating its head. The level of adult-hood presumably strongly modulates the accessibility to thedevelopment module but can also impact on the parametersof the induction module. In the context of tissue repair, thedevelopmental module would be either not available or truncated, and as a consequence, the wound healing andthe induction module would suffice to replace the missingtissue but not to regenerate a pre-existing three-dimensionstructure.

TO CONCLUDE

Homeostasis and regenerative processes rely on thecoordinated integration at the tissue level of multiple formsof cellular plasticity. Our most recent knowledge in stem cellbiology indicates that the molecular changes that supportcellular and developmental plasticity rely on epigeneticnuclear modifications.

The Hydra and planarian model systems possess uniquefeatures to study stem cell biology, maintenance of homeo-stasis and reactivation of developmental programs in adult-

hood.Bisected Hydra polyps allow for the study tissue repair (foot regeneration) as well as different routes to achieve acomplex form of regeneration, that is, head regeneration.

In Hydra, some sustained cellular adaptations required tomaintain homeostasis, that is, a massive autophagy and amoderate apoptosis, do not exhibit similar regulationsduring the regenerative response. By contrast a limitationof autophagy is required at the tip to promote cell survivalafter amputation and a wave of apoptosis that inducesproliferation of the surrounding progenitors is needed inhead-regenerating tips after mid-gastric bisection.

We propose to view animal regeneration as an adultdevelopmental process with a tripartite modular organiza-tion:the woundhealing response,the regeneration inductionmodule and the developmental module. The wound healingresponse amplifies the various forms of plasticity availableprior to injury, the regeneration induction module develops acellular remodeling that integrates these different formsof plasticity to activate the developmental module, whichappears to make use of the tools previously used duringembryogenesis or organogenesis.

The regeneration induction module, highly constrainedby the homeostatic conditions, bridges the wound healingresponse to the reactivation of a developmental program byusing one or several forms of cellular plasticity that canbe combined: proliferation of differentiated cells, dedifferen-tiation and proliferation of precursor cells, stem cell recruit-





ment, apoptosis-induced compensatory proliferation, trans-differentiation. In numerous species, but not in mammals,the regeneration induction module corresponds to the for-mation of the blastema. In amphibians, the dedifferentiationof adult somatic cells after injury does not seem to lead topluripotency in the blastema.

Five distinct forms of regeneration can be identifiedaccording to the developmental status of the regeneratingorganism: regulative, fetal–larval, juvenile, paedomorphic,adult. We assume that the importance of the regenerationinduction module inversely correlates with the intensity of the developmental processes ongoing at the time of injury.

ACKNOWLEDGMENTS

We aregrateful to Philip Newmark forhelpful discussions andtotwo anonymous reviewers for constructive comments. The work inour laboratory is funded by the Swiss National Foundation, theGeneva State, the NCCR ‘‘Frontiers in Genetics’’ Stem Cells &

Regeneration pilot project, the Claraz Donation and the AcademicSociety of Geneva.

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brigitte gallot and luiza ghila- cell plasticity in homeostasis and regeneration

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