06Crescenzi

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CHAPTER 6

Reactivation of the Cell Cycle in Terminally Differentiated Cells , edited by Marco Crescenzi.©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

Cellular DedifferentiationDuring Regeneration:The Amphibian Muscle System

Elly Tanaka

Abstract

A mphibian limb regeneration represents a striking system where the reversal of musclecell differentiation occurs in response to physiological stimuli. During this process,dedifferentiation is used to form progenitor cells for tissue repair. In response

to injury, multinucleated muscle cells resolve into mononucleate cells that undergoproliferation.

The extracellular signal that initiates S-phase re-entry from the differentiated state isa serum factor that is distinct from known polypeptide growth factors such as FGF orPDGF. The factor is activated by thrombin proteolysis thus closely linking the initiationof dedifferentiation to wound healing.

Muscle cell dedifferentiation has not been described after injury in mammals and a major question is why it does not occur. The serum activity that stimulates newt myotubes

is found in sera from all animals tested so far, yet mouse myotubes do not respond to theserum factor. Therefore there appears to be an intrinsic difference between newt andmammalian myotubes. The differences in the intracellular pathway to cell cycle re-entry lies in the retinoblastoma pathway. Serum addition stimulates retinoblastoma phosphory-lation in newt myotubes but not in mouse myotubes. It is not yet known where along thepathway the species difference lies. The different responses of newt and mouse myotubesto serum is the first discrete cellular assay that relates to the differences in regenerativeability between species.

Less is known about the molecular control of mononucleate formation from syncytialmyotubes. Formation of mononucleate cells and cell cycle re-entry are separable processesthat are not interdependent. The initial re-entry into S-phase can occur when nuclei arestill within the myosin-positive syncytium and the formation of mononucleate cells doesnot depend on cell cycle re-entry. Recently, molecular insight was gained when it was

shown that the expression of the msx1 gene could drive mouse myotubes to generatemononucleate cells. The mononucleate derivatives were able to form multiple cell typessuch as osteoclasts, chondrocytes, and adipocytes. These dramatic results additionally show



Reactivation of the Cell Cycle in Terminally Differentiated Cells 78

that the dedifferentiation program is accessible to mammalian myotubes. The application

to tissue repair is clearly an important avenue of future investigation.

Introduction

The Reversibility of Muscle Differentiation in Vertebrates Development of the skeletal muscle system involves the proliferation of multi-potential

progenitors followed by lineage restriction and finally by differentiation. During the processof terminal differentiation, the myogenic progenitor cells called myoblasts withdraw fromthe cell cycle and fuse to form syncytial myotubes and fibers that express muscle-specificgene products. This post-mitotic, differentiated phenotype is stably maintained and may be terminal with respect to the organism’s lifespan. Although it is long-lasting, thedifferentiated state is actually maintained by active cellular mechanisms.1 Cell nuclei withinmyotubes can be experimentally induced to start DNA replication again by expression of

viral oncoproteins, deletion of the retinoblastoma gene, or by fusion of muscle cells withfibroblasts indicating that the DNA replication machinery is not irreversibly repressed.2-5

Secondly, muscle-specific gene expression is propagated by the continual action of transcriptional activators. This was initially shown in principle when heterokaryons weremade by fusing differentiated myotubes with non-muscle cells. In the muscle cellenvironment the nuclei of non-muscle cells initiated muscle gene transcription.6 Muscledifferentiation therefore represents a dynamic steady state that can in principle be re-versed. This property raises the question of whether dedifferentiation—the formation of proliferating mononucleate cells from differentiated muscle cells—ever occurs under naturalconditions.

The Reversal of Muscle Differentiation During Regeneration Amphibian limb regeneration represents the most striking example where dedifferen-

tiation of vertebrate muscle occurs in a physiological context. After limb amputation skel-etal muscle cells lose their differentiated character, re-enter the cell cycle, and produceproliferating mononucleate cells from the multinucleate syncytium.7-10 In regeneration,dedifferentiation is intimately associated with perfect tissue repair and therefore the newtrepresents an important model system for studying the production of progenitor cellsfrom adult tissues. Notably this mechanism contrasts with the activation of quiescent stemcells, a well-known form of tissue repair and renewal in other vertebrates. As reviewedhere, the reversal of muscle differentiation during newt limb regeneration has been stud-ied at the cell and molecular level.9,11-13 From these studies, it is clear that reversal involvesthe response to extracellular signals induced by injury and that there are at least two paral-lel and distinct pathways that are activated in the muscle cell to execute cell dedifferentia-tion. These insights provide not only a mechanistic understanding of the process but they also represent a platform for comparing the regenerative ability of newts versus mammals

by specifically focussing on dedifferentiative ability. A provocative hypothesis that resultsfrom such studies is that an underlying difference between mammals that cannot andnewts that can regenerate complex structures such as the limb is the capacity of differenti-ated cells such as muscle to undergo dedifferentiation at the site of injury.



79 Cellular Dedifferentiation During Regeneration

Skeletal Muscle Dedifferentiation Produces Progenitor Cellsfor Limb Regeneration

The Formation of Progenitor Cells from Mature Tissue In members of the Salamandroidea (Salamander) superfamily such as the newts, limb

transection stimulates large-scale repair that perfectly replaces all the tissues in the limbincluding muscle, bone, nerve, dermis and skin (Fig. 1).14-17 After the initial sealing of the

wound by migrating epidermis, the injured tissue produces a zone of proliferating,apparently undifferentiated mesenchymal cells, called the blastema (Fig. 2A). Blastema formation takes from 5 to 14 days depending on the species and age of animal.7,8,18 Theseblastema cells along with the overlying epidermis reactivate developmental programs toreplace the missing portion of the limb.19-25

A major question since the beginning of regeneration research has been which tissuescontribute cells to the blastema and by what mechanism. Experiments tracing the originof the blastema through transplantation of marked tissues showed that the blastema de-rives from multiple tissues, including dermis, peripheral nerve, bone and muscle.26-31 Inthese classical experiments, however, the grafted tissue constituted a complex mix of dif-ferentiated cells including connective tissue and blood vessels. Therefore the transplanta-tion of tissue did not completely resolve the cellular origin and the mechanism by whichthe blastema was formed.

Descriptive Evidence for Muscle Dedifferentiation During Regeneration

Here we will focus on the contribution to the blastema of a single differentiated celltype, the multinucleated skeletal myotube, reviewing first the descriptive evidence fordedifferentiation of muscle fibers and more recent experimental evidence that multinucle-ate muscle cells dedifferentiate during regeneration. Described here are three of the classi-cal histological studies on regenerating muscle that argued particularly strongly that multi-nucleated fibers contributed cells to the blastema by budding mononucleate cells directly from mature fibers.7,32 In these studies regenerating limbs were fixed, sectioned and ob-served by light and electron microscopy at varying times after amputation during the timethat the blastema was forming. Starting 5 days post-amputation, the ends of the musclefibers having lost their myofibrillar structure appeared to bud off nuclei surrounded by a small portion of cytoplasm from the muscle syncytia (Fig. 2A).7,8 In some cases themononucleate cells contained small remnants of myofibrils revealing their origin. These“budded” cells displayed enlarged nuclei compared to nuclei within differentiated musclefibers (Fig. 2B).8 In a second experiment Hay and Fischman found a small number of multinucleated fibers at the end of the limb that had incorporated tritiated thymidine—suggesting that the nuclei of differentiated muscle cells were returning to the cell cycleprior to budding.33 These observations were interpreted as evidence of muscle

dedifferentiating to give rise to mononucleate blastema cells but the studies remainedcontroversial since the fate of single muscle cells could not be definitively followed. Theseinterpretations were open to the possibility that the observed events actually representeddifferentiation of myoblasts into new muscle fibers at the end of the limb rather than thereverse.34 Furthermore, since thin sections that only encompassed a small portion of a celldiameter were examined, it was difficult to definitively show that the mononucleate cells

were truly separated from the syncytium.




Fig. 1. Stages of newt limb regeneration. The arm on the left was amputated through the upper arm and resultsin regeneration while the arm on the right was amputated through the lower arm. In both cases only the missing

portion of the limb is regenerated through the formation of a blastema (days 7-21) and subsequent limb morpho-genesis (days 28-70). Photo series were taken at successive times (top to bottom): pre-amputation and then 7, 21,25, 28, 32, 42, 70 days post amputation. (From Goss RJ, Principles of Regeneration, 1969, p142, copyright

Academic Press, Inc.)



81Cellular Dedifferentiation During Regeneration

Fig. 2A. Section through a regenerating larval limb at 5 days ( Amblystoma punctatum). On the left is the mature

tissue, containing muscle (Mus) and nerve (Ne). The right part of the tissue is the regeneration blastema (Bl)containing mononucleate cells, and covered by the wound epidermis (Ep). G denotes an osteoclast. B. Histologicalevidence for muscle cell dedifferentiation. Enlarged view of the transition zone between the ends of muscle fibersand the blastema. N´´ , N´, and N mark what are apparently successive stages in the dedifferentiation process. N´´is an elongated nucleus characteristic of a muscle cell nucleus. N´ is an elongated but enlarged nucleus of a dedifferentiating muscle fiber. A row of dedifferentiating cells with rounded nuclei (N) are seen budding at the endsof fibers. The cytoplasm in these cells has lost the myofibrillar structures characteristic of differentiated muscle.(From E.D. Hay, Dev. Biol. 1959; 1:558, Academic Press)




Experimental Evidence for Muscle DedifferentiationMore recent experiments using fluorescently labelled cells have provided positive

experimental evidence that multinucleated muscle cells indeed reverse their differentiationto produce proliferating mononucleate cells. Lo, Allen and Brockes used myoblast culturesderived from newt limb that were induced to form myotubes in culture by lowering serumconcentrations in the media (Fig. 3).35 These cultured myotubes were separated frommononucleate cells by size-selective sieving and then labelled by microinjection of myotubes

with a fluorescent cytoplasmic lineage tracer prior to implantion into regenerating limbs.Histological sectioning of the implant-containing limbs showed that by 7 days post-implantation lineage label was in mononucleate cells of the blastema, indicating that themultinucleated myotubes had given rise to mononucleate cells. Quantitation of cell numberover time indicated that these labelled mononucleate cells proliferated in the blastema.This experiment was later confirmed using myotubes containing a retrovirally-integratedlineage marker, demonstrating that mononucleate cells did not arise from the cytoplasmic

transfer of injected lineage tracer.36

These results provided the first experimentaldemonstration that the environment of the regenerating limb induces the dedifferentia-tion of multinucleate myotubes into proliferating mononucleate cells.

The Role of Dedifferentiation in RegenerationLimb regeneration is a complex process because many cell types contribute to the

blastema. The quantitative contribution of dedifferentiated muscle to the blastema is notyet known because experiments have depended on the implantation of exogenous myotubesas indicators of dedifferentiation. Furthermore, muscle dedifferentiation has yet to be spe-cifically inhibited to test the dependence of regeneration on this process. Experimentsexamining the contribution of dermis to the regenerating limb blastema indirectly sup-port the role of muscle.28 Local X-irradiation of the limb inhibits regeneration by inhibit-ing cell division. A cuff of unirradiated skin including epidermis and dermis was trans-

planted onto an irradiated limb resulting in limbs where the skin was unirradiated butinternal tissues including muscle were irradiated. When such limbs were amputated throughthe grafted skin and allowed to regenerate, the skin, bone, and ligaments were regenerated

with perfect skeletal patterning. The regenerated bone and ligaments derived from thegrafted skin. Muscle, however, was largely missing. This result implies that muscle tissuemakes a necessary contribution to the blastema, although other interpretations are pos-sible. As with other tissue grafting experiments, these observations also did not provideinsight into whether the contribution of muscle would occur through dedifferentiation oractivation of a resident progenitor cell in muscle. An experimental resolution to theseissues would be to transplant purified myotubes into the X-irradiated limbs with skingrafts to determine if implantation of myotubes rescues the lack of muscle.

The importance of dedifferentiation to regeneration is best seen in simpler structuresother than the limb. In salamanders, a number of the eye tissues can be regenerated.37 Forexample removal of the lens of the eye induces its regeneration from the dorsal pigmentediris epithelium to which the lens was originally attached. In this case a single cell type, theretinal pigment epithelial cell (RPE) clearly gives rise to the cells of the regenerating lensthrough dedifferentiation and transdifferentiation. This RPE-to-lens transition has beenrigorously demonstrated in clonal cell culture of embryonic chick RPE and occurs in twodistinct steps where RPE cells lose their differentiated features and begin to proliferatebefore the second step of transdifferentiating into lens.38




Reversals of muscle differentiation are also known to occur during regeneration in

other, evolutionarily distinct organisms. The invertebrate hydrozoan jellyfish (Podocoryne carnea ) is capable of undergoing extensive regeneration that involves transdifferentiationof skeletal muscle into other cell types.39,40 This transition has been analysed in cell culture.

When a homogeneous sheet of mononucleated striated body muscle cells was treated withthe proteases pronase or collagenase, digesting the basement membrane, the muscle cellsunderwent DNA synthesis, proliferation and transdifferentiation giving rise to both smoothmuscle and RF-amide positive neurosecretory cells.41,42 Interestingly the transition from

Fig. 3. Experimental evidence for myotube dedifferentiation during regeneration. Myotubes were formed inculture from newt myoblasts and then purified by passing through sieves. Myotubes were selectively marked by microinjection of fluorescent lineage tracer and subsequently pelleted and implanted into 5-day regenerating limbs. Limbs were allowed to heal and regenerate for varying amounts of time before sectioning and examinationfor the profile of lineage tracer. After 7 days fluorescent cells were found in mononucleate cells of the blastema andthe number of cells increased with time. (Adapted from Lo et al. PNAS 1993; 90:7231)




skeletal to smooth muscle did not require cell cycle progression, while transdifferentiation

to the neurendocrine lineage did.43

Analysis of the transdifferentiation event in jellyfish indicates that changes in themuscle cells´ interaction with the extracellular matrix induce the destabilization of thedifferentiated phenotype. Cells that maintained a rigid attachment to the ECM remainedstably differentiated while those that were loosened and did not maintain a spread or“stretched” state underwent cell cycle re-entry and transdifferentiation. Transdifferentiation

was observed when pieces of striated muscle that had not been treated with protease –called “mechanically isolated pieces”—were plated onto a new piece of extracellular matrix.44

When the muscle cells were plated onto ECM that was firmly adherent to glass thosemuscle cells straddling the junction between the new and old ECM underwent DNA synthesis and transdifferentiation into neurons. These cells had apparently lost contact

with both ECMs, and did not have a spread morphology. In contrast, those cells that hadmoved beyond the junction and had adopted a spread or stretched state on the new ECM

did not undergo proliferation and transdifferentiation. To test the role of cell tension intriggering transdifferentiation, pieces of mechanically isolated striated muscle cells werecombined with floating pieces of ECM where cells would migrate onto the new ECM, butthey could not adopt a spread morphology and generate tension across their cell diameters.In this case cells throughout the explant underwent DNA synthesis and transdifferentiationinto neurosecretory cells.44 In the jellyfish, the ability to analyse transdifferentiation of a homogeneous sheet of muscle cells reveals the important role that the reversal of differentiation plays in providing progenitor cells for regeneration. In the future it will befascinating to determine whether the signalling processes controlling dedifferentiation ininvertebrate muscle cells have significant parallels with dedifferentiation of newt muscle.

Muscle Progenitor Cells During Repair: Muscle DedifferentiationVersus Satellite Cells

In vertebrates such as mammals and birds, adult skeletal muscle contains a dormantpopulation of mononucleate progenitor cells called satellite cells that lie between the dif-ferentiated muscle cell and the basal lamina.45,46 In growing animals these cells contributeto muscle tissue growth by proliferating and then fusing with resident fibers.47 In adults,injury stimulates the satellite cells to divide and to later fuse into fibers.48 In essence satel-lite cells represent a muscle-specific stem cell. Recent evidence also indicates that cells of endothelial origin can also act as muscle progenitor cells.49

Are muscle satellite cells or other myogenic progenitors also activated during newtlimb regeneration? Adult newt muscle apparently does not have classically defined satellitecells.50 However whether other interstitial mononucleate cells that reside within the maturemuscle are stimulated to produce myogenic cells for regeneration is still an open question.Schrag and Cameron argued that the outgrowth in culture of mononucleate, myogeniccells from urodele limb muscle explants represented the migration and proliferation of a mononucleate precursor population residing between muscle fibers because the authorscould not see evidence of dedifferentiating muscle fibers in their preparations.51 In theseexperiments, however, the origin of the dividing cells in culture was not determined so itcould not be ruled out that muscle fibers had actually dedifferentiated, losing their well-defined morphology and making them difficult to identify.

It is clear however that in salamanders muscle dedifferentiation occurs in tissues wheremyogenic progenitor cells are still present. Hay´s histological work on limb regeneration8

and our own recent work tracing endogenous muscle cell fate during tail regeneration51A




indicate that muscle dedifferentiation occurs during regeneration in larval salamanders

where rapid overall body growth is still occurring, and where mononucleate muscle pre-cursors must still be actively contributing to growing muscle. This means that in someparts of the tail close to the site of injury multinucleate muscle cells are dedifferentiating toform the regeneration blastema while in the non-injured tissue myoblasts are fusing intogrowing fibers. This poses the interesting question why dedifferentiation is invoked at a stage where myoblast proliferation is clearly still occuring. Recent work described in thelast section of this Chapter indicates that the progenitors produced from dedifferentiationare more multipotent than myoblasts. This suggests that dedifferentiation may be requiredduring regeneration to produce flexible cell types that can be influenced by extracellularsignals to form many tissue types.

Regulatory Pathways Leading to Dedifferentiation What are the mechanisms that regulate newt muscle cell dedifferentiation? Muscle

dedifferentiation is a complex process involving dramatic changes in cell architecture as well as proliferative potential. Differentiated muscle cells contain highly organizedactomyosin arrays that constitute the contractile apparatus. During regeneration, thismyofibrillar structure is apparently broken down within the dedifferentiating muscle cell.7,8

Single nuclei leave the syncytium to form mononucleate cells. While undergoing thisgross morphological change these mononucleate cells have overcome cell cycle withdrawaland regain their proliferative potential. The analysis of cultured newt myotubes has provideda means to analyze in depth the cellular and molecular regulation of dedifferentiation. Inparticular, recent experiments have revealed that the cell cycle re-entry and the morpho-logical changes related to dedifferentiation are two independent processes. S-phase re-entry can occur in multinucleate, myosin positive myotubes before complete breakdownof myofibrils occurs.10,33 On the other hand, fission of myotubes into mononucleate cellsdoes not depend on S-phase re-entry or cell cycle progression within the myotube.13

Intracellular Regulation of Cell Cycle Re-entry from the Differentiated State

Newt Myotube Nuclei Undergo S-Phase in Response to SerumTanaka et al showed that newt myotubes, when purified by sieving and replated in

culture could be stimulated by serum to undergo a complete S-phase. 10 Myosin heavy chain staining remained high for at least 8 days indicating that under these circumstancesmyofibrils do not break down. In culture the myotubes arrested in G2 phase and noformation of mononucleate cells was ever observed. Therefore culture conditionsreconstituted one step of the dedifferentiation process. The S-phase re-entry observed inculture likely reflects the initial steps of dedifferentiation that occur in vivo. Tritiated thy-midine labelling of regenerating limbs resulted in incorporation of label into nuclei within

multinucleate muscle fibers at the transition zone between mature tissue and blastema.33

In contrast, mouse myotubes, once differentiated, do not enter S-phase in response toserum factors.52 We hypothesize that the ability to re-enter the cell cycle from the differen-tiated state is a fundamental basis for the ability to establish progenitor cells that willundertake complex regenerative events. Therefore the newt muscle cell cycle re-entry rep-resents a foothold into the analysis of regenerative ability on the cellular and molecularlevel. In our view it is of great interest to understand the differences in cell signalling and




cell differentiation that underlie newt myotubes that participate in regeneration, and mouse

myotubes that do not.

Mouse Myotube Nuclei Do Not Undergo S-Phase UnlessComponents of the Retinoblastoma Pathway are Inactivated

In newt myotubes S-phase re-entry in response to serum occurs without “unnatural”intervention. In contrast when wild-type mouse myoblasts are put into low serum they enter G0 and fuse to form myotubes that cannot be induced by growth factors or serum tore-enter S-phase.52 However, mouse myotube nuclei will undergo serum dependent S-phase when key cell cycle regulators are perturbed either via genomic deletion, viral pro-tein expression or parasite infection.2-4,53,54 The work forcing S-phase re-entry in mousemyotubes provided molecular clues about the intracellular pathway that is activated innewt muscle. In particular the mouse studies showed that the retinoblastoma (Rb) proteincontinually represses the cell cycle in postmitotic myotubes. Inactivation of Rb either via

viral proteins such as large T antigen or by genomic deletion results in serum dependent S-phase in mouse myotube nuclei. Rb is a central regulator of the cell cycle that repressestranscription of cell cycle progression genes by inhibiting the E2F family of transcriptionalactivators via direct binding and by recruiting histone deacetylase to the complexes.55-61

Conversely Rb promotes expression of genes related to cell differentiation.53,62-64

The Role of Rb in Cell Cycle Re-entry The retinoblastoma protein is a negative regulator of entry into S-phase in all cells. In

proliferating cells such as fibroblasts, growth factor signalling induces the phosphorylationof Rb at the G1 to S transition. This phosphorylation inactivates Rb and thereby allowsprogression into the cell cycle (Fig. 4).65 The kinases involved in phosphorylating Rb arethe cyclin-dependent-kinases that control the timing of cell cycle transitions.66 In particu-lar, the cyclinD/cdk4 heterodimer is a serum-responsive kinase that phosphorylates Rb

and mediates the growth factor stimulation of cell proliferation. The cyclinE/cdk2 kinasealso phosphorylates Rb and promotes the G1-S transition.

During muscle differentiation the regulation of Rb protein phosphorylation by growthfactors is shut down. In wild-type mouse myotubes that are stably withdrawn from the cellcycle the Rb protein is no longer phosphorylated in response to serum (Fig. 4). 2 Many growth factor receptors are down regulated upon myogenic differentiation, making themuscle cell “blind” to those extracellular stimuli. However, during differentiation musclecells become refractory to growth factor stimulation well before down-regulation of receptorsindicating the action of an internal inhibitor of cell cycle re-entry.67 For example, in theearly stages of differentiation mouse myotubes can still induce the early response genes

when challenged with serum but other components of the cell cycle re-entry machinery remain repressed and the cells remain out of the cell cycle.68,69 This internal inhibition tocell cycle re-entry is attributable largely to cyclin-dependent-kinase inhibitors (CKIs).

Regulation of Rb Phosphorylation by CKIsThe CKIs are likely to be the predominant inhibitors of Rb phosphorylation and thus

cell cycle entry in muscle cells. They are proteins that bind cyclin-dependent-kinases inthe nM range and inhibit their kinase activity. There exist two general families of CKIs.70

The P21CIP1 family includes p21CIP1 , p27KIP1 and p57KIP2. These proteins bind and in-hibit all cyclin-dependent kinases that act at the G1-S transition, including cyclinD/CDK4




and cyclinE/CDK2 (Fig. 4). On the other hand the p16 INK4 family consists of p15INK4A , p16INK4B, p18INK4C and p19INK4D. These proteins bind and inactivateCDK4/6 specifically.

Fig. 4. The retinoblastoma pathway in cycling cells versus differentiated myotubes. In G1, hypophosphorylatedRb actively prevents E2F from initiating transcription of S-phase genes. As a cycling cell passes through the G1to S transition, Rb is phosphorylated by cyclinD/CDK4 and then cyclinE/cdk2, rendering Rb unable to bind E2F.E2F is now able to initiate gene transcription. In myotubes that have withdrawn from the cell cycle Rb maintains

its repressive function through the presence of p21CIP1 family members and p18INK4C family members that preventthe cyclin-dependent kinases from phosphorylating Rb.




In the process of muscle differentiation several CKIs are highly induced in a distinct

temporal progression. P21CIP1 is upregulated early upon serum withdrawal, under thecontrol of myogenic factors such as MyoD.71-73 The relatives of p21, p27 KIP1 and p57KIP2

are then upregulated.74,75 In later stages p18INK4C, is highly expressed.76 In mouse myotubes,once differentiated, the inhibitory action of CKIs is apparently refractory to serum stimu-lation. Furthermore the expression of multiple CKIs during differentiation results in a situation where maintenance of cell cycle withdrawal is apparently not dependent on any one CKI. This is evidenced by the apparently normal muscle differentiation observed inp21 and p27 knockout mice.70,77-82 However, the importance of these inhibitory proteinsin muscle cell cycle arrest has been demonstrated in several ways. Myotubes in mice thatare doubly mutant for p57 and p21 undergo cell cycle re-entry similarly to cells from theRb-/- mice.75 Second E1A, a viral oncoprotein that binds to both Rb and p21 causes cellcycle re-entry in mouse myotubes. Mutational analysis of E1A functional domains indi-cates that the ability to neutralize p21 as well as pRb is required for inducing cell

cycle re-entry.83

Does Rb Have a Role in Newt Myotube Cell Cycle Re-entry?How then do newt cells achieve cell cycle re-entry from the differentiated state? First,

it should be noted that newt myotubes are normally as firmly differentiated as wild-typemouse myotubes. For example in Rb-/- or p27 -/-;p57 -/- mice muscle tissue forms but

widespread BrdU incorporation in muscle fibers and other pathology is observed.75,84 Incontrast, newt muscle is stably withdrawn from the cell cycle in the uninjured animalindicating that the differentiation machinery is completely intact. Exposure of newtmyotubes to injury (in vivo) or serum (in vitro) results in cell cycle re-entry in a highly controlled manner. Tanaka et al showed that newt myotubes unlike mouse myotubes canphosphorylate Rb in response to serum (Fig. 5).10 The functional importance of Rbphosphorylation in newt myotube cell cycle re-entry was demonstrated by expression of

an unphosphorylatable form of Rb in the newt myotubes that dominantly inhibited S-phase re-entry. These results indicated that somehow in the newt myotubes cyclin-dependent-kinases became active in response to serum. This latter conclusion was confirmedby showing that the forced expression of the human CKI p16 INK4a in the newt myotubesefficiently blocked the cell cycle response to serum. The specificity of p16 for CDK4/6indicates that serum-induced activation of the CDK4/cyclinD kinase is important for cellcycle re-entry in newt myotubes.

Regulators of the Rb Pathway Play an Important Role in Newt Myotube Cell Cycle Re-entry

It is not yet known how the cyclinD/cdk4 pathway is activated in the newt myotubessince none of the components of the pathway have been isolated or studied. Two generalclasses of models can be proposed to explain the different response to serum of newt and

mouse myotubes. In the first model the newt myotubes may be lacking a factor present inmouse myotubes that makes cell cycle arrest permanent. An example of the first class would be if one of the several CKIs that is not necessary for cell cycle withdrawal but isrequired for permanence of cell cycle arrest is not expressed in the newt myotubes. p18INK4C

which in mouse myotubes is upregulated in the late stages of differentiation well after cellcycle withdrawal has already taken place could be such a molecule.76 There is no data yetfrom genomic deletion in mouse to test this hypothesis. It is interesting, however, that




Fig. 5. Newt myotubes phosphorylate Rb in response to serum. Newt myotubes maintained in low serum are withdrawn from the cell cycle and contain hypophosphorylated Rb (lane 1). Newt myotubes stimulated withserum phosphorylate Rb, as evidenced by gel mobility shift (lane 2) and enter S-phase. Proliferating myoblasts alsocontain phosphorylated Rb (lane 3).




mice lacking p16INK4A , a close relative of p18 that is expressed in complementary tissues,

develop normally but are prone to tumors. Furthermore fibroblasts derived from p16-/-

mice do not undergo cellular senescence—another case of permanent of cell cycle arrest—despite accumulation of another CKI, p21, over time.85 Intriguingly, primary newt myo-blasts and blastema cells are immortal and do not display cellular senescence supporting the possibility of differences in regulation of INK4 family members in the newt. Thecloning of the newt CKIs has not yet been achieved most likely because of high sequencedivergence. Data from the sole Xenopus p28 XIC gene, which displays hybrid features of both p27 and p21, indicates that the CKIs have evolved rapidly.86,87 These observationssuggest that the cyclinD/CDK4 pathway, in particular the CKIs, may be a fulcrum forevolutionary changes that modulate the features of cell cycle arrest.

In the second class of models the newt and mouse cells may differentiate along iden-tical pathways but newt cells upon serum stimulation may be able to activate a signalling pathway that “dominantly” stimulates cell cycle re-entry from the differentiated state.

Mammalian myotubes may lack this pathway. This dominant pathway might take severalforms. For example, many of the CKIs are known to be targets of controlled proteindegradation and this pathway might be activated in the newt cells but not in mouse cells.88,89

Another possibility is the induction of CDK4 expression, which is not induced by serumin mouse myotubes at high enough levels to overcome the inhibitory CKIs.69 The differ-ence in ability between newt and mouse myotubes to activate a “dominant” pathway thatovercomes the cell cycle block in differentiated cells could be due either to a limitation inan intracellular component, or the mouse myotubes may merely be lacking the receptorfor the serum factor that stimulates the newt myotubes.

Clearly the pathway from extracellular signal to Rb will need to be understood toexplain the difference between newt and mammalian cells in their serum responsiveness.In addition to understanding the differences in the intracellular signalling pathway, theidentity of the extracellular signal that stimulates the newt cells is of particular interest. Asmentioned previously, many growth factor receptors are down regulated upon differentiationof mouse myotubes. As discussed in the next section the newt myotubes, upondifferentiation, acquire responsiveness to the factor within serum that stimulates myotubeS-phase. This serum factor is unable to stimulate newt myoblasts, indicating that theserum factor is distinct from conventional myoblast mitogens such as EGF and FGF.

Extracellular Signals Initiating the Cell Cycle Derive from Wound Healing Responses

The Loss of Cell-Cell Contact is Required for Newt Myotubes´Responsiveness to Serum

After amputation, major changes in the limb tissue occur. The blood clots, cells contractand cell-cell contacts are disrupted. Later ECM is degraded and the wound epithelium

migrates over the exposed tissue.14

Analysis of S-phase re-entry in cultured newt myotubesdemonstrated that wound-healing signals play a major role in cell cycle control. First Tanaka et al found that myotubes would only re-enter S-phase when they were not contact inhib-ited, suggesting that in vivo muscle fibers must be released from contact with their neigh-bors in order to re-enter S-phase.10 This feature of cell cycle re-entry in the newt myotubes is notable, since newt myoblasts and blastema cells have the unusual property of not being contact inhibited.




The Clotting Cascade Activates a Serum Factor That StimulatesS-Phase Re-entry

Serum is the soluble component of blood formed after clotting. The cell cycle stimu-latory activity in serum has several interesting properties that indicate that it is regulatedby clotting.12,90 Here, we will refer to the factor in serum that stimulates newt myotubes as“S-phase re-entry factor” (SPRF). Low serum conditions (1% serum) normally do notelicit the S-phase response in the newt myotubes while high serum concentrations (10%)do. However, addition of thrombin to low serum media induced S-phase re-entry in thenewt myotubes. One possibility was that thrombin was SPRF—it directly stimulated a cell surface receptor for thrombin on the newt myotubes to induce re-entry. The existence

of a G-protein coupled receptor for thrombin made this possibility particulary appeal-ing.78,91 However, it was shown that thrombin is not SPRF itself but rather, thrombincleaves a component in the low serum media that results in the generation of SPRF activ-ity (Fig. 6).90 This was demonstrated by pre-treating low serum media with thrombin for24 hours and subsequently inhibiting all thrombin protease activity before addition of thepreparation to cells. Such preparations contained SPRF activity whereas preparations wherethrombin and inhibitor were added simultaneously did not contain SPRF activity.

Fig. 6. Thrombin treatment of low serum media generates SPRF activity. A. Experimental protocol. Low serummedia was incubated with thrombin for 24 hours and then proteolytic activity was inhibited with hirudin or

PPACK. These samples stimulate newt myotube S-phase (B. solid line). In contrast, if thrombin and inhibitor areadded at the same time, no stimulation of S-phase is observed (B. dotted line).




The involvement of thrombin in SPRF biogenesis uncovers the role of blood clotting in triggering dedifferentiation. Clotting is a highly localized process that is initiated by tissue injury.92 Thrombin´s main role in blood is the terminal protease in the clotting cascade that results in the cleavage of fibrinogen to fibrin (see later, Fig. 8). After cleavagefibrin self-associates to form an insoluble fibrous network which forms the structural basisfor the clot. Though it cleaves fibrin at relatively specific protein sequences, thrombin alsohas multiple other substrates in serum. Fibrin, or its degradation products do not haveSPRF activity and so far, none of the other known, direct substrates of thrombin such as

ApoE or thrombospondin has proven to be SPRF (E. Tanaka and J. Brockes, unpub-lished). It is possible that SPRF represents an unknown substrate of thrombin. Alterna-tively thrombin does not cleave SPRF directly but rather an upstream regulator of SPRF.

Purification of S-Phase Re-entry FactorThe identity of the SPRF activity that is downstream of thrombin proteolysis and

acts directly on newt myotubes is critical to understanding cell cycle re-entry from thedifferentiated state. The direct SPRF activity was found to be significantly enriched incommercially available crude thrombin preparations.90 These preparations have provedcritical to the further characterization of the SPRF activity. The activity present in total

Fig. 7. Fractionation of crude thrombin reveals that SPRF stimulates S-phase in myotubes but not mononucleatemyoblasts. Addition of total serum or crude thrombin preparation to cells induces DNA synthesis in myotubesand myoblasts. The flow through fraction of crude thrombin applied to Q-sepharose contains myoblast stimula-

tory activity while the high salt elution (peakII) contains myotube stimulatory activity.




fetal calf serum and in the crude thrombin preparation has an apparent molecular weightof 200,000 Daltons. Fractionation of crude thrombin on strong cation exchange resinsseparates thrombin, an indirect activator of S-phase re-entry, from SPRF which directly acts on newt myotubes. Direct (SPRF) versus indirect (e.g., thrombin) activities can bedistinguished by the requirement of low serum in the assay.12,90 Thrombin, which gener-ates SPRF from serum always requires the presence of low levels of serum in the media asa substrate. SPRF present in crude thrombin can act in the complete absence of serum inthe media.

Clearly it will be important to identify the polypeptide sequence of SPRF in order tounderstand how it stimulates cell cycle re-entry in newt myotubes but not mouse myotubes.Fractionation of crude thrombin on cation exchange followed by anion exchange, and

finally affinity to heparin sulfate has been used to purify SPRF 2000-fold over serum. Thispurification scheme yields a protein preparation of 10 μg/ml with 15-20 bands visible by silver staining (E.Tanaka, D. Drechsel, J. Brockes unpublished). Given a molecular weightof 200 kD, if SPRF were to represent one of the visible bands, it would be active at a concentration of 1-5 nM. Interestingly, this represents a potency similar to those of theanti-angiogenic factors endostatin and angiostatin.93,94 These two molecules represent goodparadigms for SPRF, as they are cleavage products of serum and extracellular matrix pro-teins that have bioactive properties different from the parent molecule.

Fig. 8. Signals leading to SPRF generation and myotube cell cycle re-entry. Limb amputation results in multiple wounding responses, one of which is the blood clotting cascade (black). This cascade results in the activation of the protease thrombin that cleaves fibrinogen into fibrin. Thrombin proteolysis also results (directly or indirectly)in the activation of SPRF from an inactive precursor form. This active SPRF elicits cell cycle re-entry in newtmyotubes that participate in regeneration. SPRF does not stimulate S-phase in newt myoblasts. SPRF also does

not stimulate cell cycle re-entry of mouse myotubes that do not participate in complex regeneration.




The S-Phase Re-entry Factor is Distinct from Conventional Myoblast

Growth FactorsBased on partial purification of SPRF, it can be shown that it is distinguishable from

classic peptide growth factors in several ways. First, its high molecular weight is characteristic.Second, fractionation of crude thrombin on Q-sepharose yields a preparation that stimu-lates myotube S-phase but not myoblast S-phase (Fig. 7).90 The SPRF activity present inthrombin-treated serum also displays specificity for myotubes over myoblasts (E.Tanaka,D. Dreschsel, J. Brockes unpublished). This characteristic of SPRF strongly contrasts withpeptide growth factors such as FGF, EGF, PDGF, and IGF, all of which stimulate the newtmyoblast cell cycle but not myotubes. The specificity of SPRF to myotubes means thatresponsiveness to SPRF is acquired upon differentiation at the same time that newt myotubes(and mouse myotubes) are losing responsiveness to classic peptide growth factors.

Is SPRF a “Dedifferentiation” Factor?It is not yet known why mammalian skeletal myotubes cannot respond to SPRF. In

other words, it is not yet known if mouse myotubes also express the receptor for SPRF onthe cell surface but differ in their internal cell cycle circuitry or whether the only limitationin the mouse response is the lack of cellular receptor for SPRF. Interestingly, Rb-/- mousemyotube cell cycle re-entry requires the presence of serum. It has not yet been possible totest whether this serum stimulation is due to SPRF or to other, more conventional growth factors.

SPRF activity is found in serum from animals ranging from chicken to human(E.Tanaka, J. Brockes unpublished). Therefore it likely represents a molecule which normally has another function in all animals and has been coopted for dedifferentiation in the newt.

An intriguing possibility is that SPRF is used in other animals during other contexts of tissue repair such as hepatocyte proliferation during liver repair. This notion is still to be tested.

Figure 8 summarizes the properties of SPRF and its relationship to wounding includ-ing its activation by thrombin and its specificity for newt myotubes. Both the contact

inhibition of S-phase re-entry and stimulation by clotting provide appealing physiologicalrationales for the local activation of dedifferentiation. The SPRF-dependent cell cycle re-entry also represents the first example of a discrete cellular difference between newt andmouse cells, analysed at the molecular level, that correlates with regenerative capacity. Thefurther analysis of these pathways promises a deeper understanding for the basis of thisdifference. Finally, the ability of SPRF to selectively stimulate myotubes but not myoblastsraises the possibility that SPRF triggers a pathway to cell cycle re-entry from the differen-tiated state that is different from that used to stimulate S-phase in normal cycling cells.

Generation of Mononucleate Cells from a Multinucleate Syncytium

The Formation of Mononucleate Cells from Myotubes Does Not Require Passage Through S-Phase

Another dimension of skeletal muscle dedifferentiation is the breakdown of cellularfeatures associated with differentiation—most notably the formation of mononucleatecells from a multinucleate syncytium. Histological studies described this as the budding of cells off the fiber.7,8 One natural question is to what extent the cell cycle re-entry is linkedto the process of mononucleate cell formation. In the mouse, interference with Rb andother pathways sometimes results in mitotic figures inside myotubes.4,5,95 In most cases




mitosis is followed by apoptosis but reports of some viable cells resulting from these mi-

totic events indicate the feasibility of such a mechanism. However, recent experiments innewt cells show that the formation of mononucleate cells from the multinucleate myotubedoes not require cell cycle progression.13 Newt myotubes were arrested before S-phaseeither by X-irradiation which activates DNA-damage-induced cell cycle arrest or by ex-pression of human p16INK4A in myotubes. When such myotubes were implanted into a regenerating limb, mononucleate cells were formed indicating that cell cycle progression

was not required for myotube fission. The persistence of the cell cycle block during theentire process of myotube fission was confirmed in two ways. First, parallel cultures invitro were shown to be unresponsive to serum throughout the duration of the experiment.Second, regenerating limbs containing implanted myotubes were labelled with BrdU.Mononucleate cells derived from arrested cells did not take up label whereas mononucleatecells derived from normal myotubes showed labelling.

The Drug Myoseverin Induces Mouse Myotubes to FormMononucleate CellsThese results suggest that intact nuclei bud off from multinucleate syncytia. As with

cell cycle re-entry, work on mouse myotubes may provide a clue to the mechanisms of myotube fission. Myoseverin, a derivative of a purine-based synthetic chemical library,caused multinucleated mouse C2C12 myotubes to resolve into mononucleate, myosin-positive cells.96 The similarity to the newt myotube phenotype was striking. Cells frommyoseverin-treated cultures incorporated BrdU and formed a higher number of colony forming units compared to non-treated cultures, providing evidence that the C2C12 cellsmay become proliferative after fission although some ambiguity remains concerning thislast issue since the myotube cultures probably contained significant numbers of unfusedmononucleate cells. Myoseverin was shown to bind microtubules and it was proposed thatdisruption of the microtubule network caused the severing phenotype. The apparent ability

of another microtubule drug, taxol, to induce myotube fission supports this view. Screening of DNA chips with RNA from myoseverin-treated cells also revealed the upregulation of many genes involved in wound-healing and cell cycle regulation. It is not yet clear to whatextent this transcriptional response is also required for the severing phenotype.

It is yet unknown if myoseverin is activating a pathway for mononucleate cell forma-tion that is normally used in the newt cells. The hypothesis that myoseverin works via microtubule disruption would predict that the microtubule cytoskeleton in dedifferentiating newt myotubes would be altered during the process and that blocking changes in microtu-bules would inhibit newt myotube fission. Myoseverin acts within 24 hours in C2C12myotubes and fission of endogenous salamander myotubes takes 3-5 days. The timing iscompatible with myoseverin activating a similar pathway to the endogenous one in newtcells. It will of course be fascinating to determine if myoseverin-treatment causes the for-mation of mononucleate C2C12 cells that have an equivalent proliferative and lineagepotential as dedifferentiated newt cells. The results from newts suggesting that the cellcycle re-entry and fission are likely independent events casts doubt on whether themyoseverin-treated C2C12 cells will have true proliferative potential. However, the possi-bility that the process of myotube fission promotes the ability to re-enter the cell cycle hasnot been ruled out.




Msx1 Expression in Mouse Myotubes Induces Mononucleate Cell

Formation and ProliferationRecently important molecular insight into the dedifferentiation process was gained

through ectopic expression of the msx1 homeobox gene in C2C12 myotubes. Msx1 is a homeobox containing gene that when ectopically expressed can prevent myoblastdifferentiation.97 During embryogenesis msx1 is expressed at the growing end of the limb

where the proliferating, undifferentiated cells reside while it is upregulated during regen-eration in newts and fish.98-102 The expression of msx1 also correlates with the ability of post-natal mice to regenerate fingertips.103

Odelberg et al expressed an inducible form of msx1 in C2C12 myotubes that hadbeen placed in growth factor rich media and followed the behavior of these myotubes overtime.104 9% of the myotubes fragmented into smaller myotubes or budded mononucleatecells. In 5% of cases myotubes generated proliferating mononucleate cells. During thededifferentiation process myotubes lost expression first of myogenin and MRF4, then

p21CIP1 and myoD. This loss-of-expression profile is the reverse sequence to the differen-tiation process. Clones derived from mononucleate cells that had budded from msx1-expressing myotubes showed a multipotency that is not observed in normal C2C12 myo-blasts. When put into the appropriate inducing media, msx1-derived mononucleate cells

were capable of forming chondrogenic, osteogenic, or adipogenic cells. This plasticity wasalso observed in C2C12 myoblasts that had transiently expressed the msx1 gene. There-fore the multipotency and the budding process are in some sense separable.

These results indicate that under the correct conditions mammalian myotubes arecapable of dedifferentiating like the newt myotubes. The msx1-induced dedifferentiationrequired serum-containing growth media. It will be interesting to determine if the factorin serum required for msx1-dependent dedifferentiation is SPRF. Although in the C2C12cells, msx1 expression was forced, in the newt, an extracellular signal produced by amputa-tion presumably elicits msx1 expression and dedifferentiation during regeneration. The

identity of the signal that induces msx1 expression in newts and fish and whether thissignal can induce msx1 expression in mammalian myotubes will be an important futureaspect of the problem. In fish, FGF-signalling expression modulates msx expression dur-ing fin regeneration.105 It is unlikely that FGFs are sufficient to initiate msx1 expression inmyotubes since FGFs are unable to induce cell cycle re-entry in newt myotubes. It willfurthermore be interesting to know if msx1 can induce dedifferentiation of other mamma-lian differentiated cell types or if it is limited to myogenic cells.

Summary and PerspectiveSalamander muscle is a striking example of differentiation being subverted by the

physiological stimulus of injury in order to produce progenitor cells for tissue repair. Theassociation with perfect regeneration raises the possibility that dedifferentiation is a criti-cal component of the salamander´s complex regenerative ability that surpasses all other

vertebrates. Dedifferentiation may well be a key aspect of the mechanisms for nuclearreprogramming that must occur to re-initiate developmental programs in the injured tissue.Such issues cannot be further investigated until more is known about the specific factorsthat initiate dedifferentiation and the ultimate potential and fate of the dedifferentiatedcells. The molecular analysis of dedifferentiation is still in its infancy and will depend onefforts to develop molecular tools and functional assays for studying regeneration.




The interplay between information from mammalian muscle and salamander muscle

has been crucial for demonstrating the possibility of reversion, and for analyzing the cellularand molecular aspects of dedifferentiation in the salamander cells. The ability to pushmammalian muscle by experimental means towards states resembling newt cell dediffer-entiation has shown that it may be possible to produce viable progenitor cells from mam-malian muscle cells. A question for the future is whether such cells will have unique prop-erties for tissue repair.

AcknowledgementsI would like to thank Chung Pin Teo for discussion of papers and David Drechsel for

comments on the manuscript.

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