Proc. Natl. Acad. Sci. USAVol. 90, pp. 7230-7234, August 1993Developmental Biology

Reversal of muscle differentiation during urodele limb regenerationDONALD C. Lo*, FRANCESCA ALLEN, AND JEREMY P. BROCKEStLudwig Institute for Cancer Research and Department of Biochemistry and Molecular Biology, University College London, 91 Riding House Street, LondonWlP 8BT, United Kingdom

Communicated by Dale Purves, May 3, 1993 (received for review March 11, 1993)

ABSTRACT Recent studies suggest that maintenance ofthe differentiated state requires continuous regulation. Limbregeneration in urodele amphibians provides a context in whichto address this issue, as limb regeneration may involve thededifferentiation of multinucleate myotubes to yield mononu-cleate blastemal cells, which then proliferate and contribute toregenerate tissues. To evaluate this possibility, cultured newtlimb myotubes were selectively microinjected with the lineagetracer rhodamine-dextran and introduced into regeneratinglimbs. In culture, such labeled myotubes were stable after 6-8weeks, and transfer of the tracer to mononucleate cells was notobserved. In contrast, after implantation of labeled myotubesunder the wound epidermis of limb blastemas, strongly labeledmononucleate cells were observed after 1 week. These cellscould be double-labeled with the cytoplasmic lineage tracer and[3H]thymidine that had been incorporated into the nuclei ofimplanted myotubes. The number of labeled mononucleatecells increased significantly by 2-3 weeks after implantation,indicating that these cells proliferated. Although the fate ofthese cells at later times was uncertain, we provide evidenceconsistent with their subsequent differentiation. These resultsdemonstrate reversal in the mononucleate-to-multinucleatetransition of vertebrate myogenesis.

Following the isolation of several myogenic genes (1-4),muscle has become a key system for investigating the controland maintenance of the differentiated state. The fusion ofmononucleate myoblasts to form multinucleate, postmitoticsyncytial myotubes is one of the most striking examples ofterminal differentiation. In understanding the mechanismsthat underlie the stability ofdifferentiation, the reversal oftheprocess, termed dedifferentiation, or a switch to anotherlineage, referred to as transdifferentiation (5), are of partic-ular interest. In a recent study it has been demonstrated thatexpression of simian virus 40 large tumor antigen can inducenuclei in cultured mouse myotubes to reenter the cell cycleand, furthermore, that this observation and the normal cellcycle arrest involved in myogenesis may reflect changes inactivation of the retinoblastoma gene product (6). Althoughthese experiments suggest the possibility of reversing myo-genic differentiation, cytokinesis of myotubes to yield mono-nucleate progeny was not reported.One natural context in which dedifferentiation may occur

is during limb regeneration of urodele amphibians such as thenewt and axolotl. Blastemal cells, the mesenchymal progen-itors of the regenerate, arise locally at the amputation planeand may derive in part from dedifferentiation ofstump tissuessuch as cartilage (7) and muscle (8-10), although the contri-bution of multinucleate myofibers remains uncertain (11, 12).To assess the reversibility of the mononucleate-to-multi-nucleate transition in urodeles, we induced myotube forma-tion in cultured newt limb cells and selectively labeled suchmyotubes by microinjection of a nontransferable, cell-impermeant lineage tracer. The labeled myotubes are stable

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in long-term culture, but after they are implanted into hind-limb blastemas a significant proportion undergo dedifferen-tiation to yield proliferative mononucleate blastemal cells.These experiments provide a direct demonstration of thereversal of muscle differentiation and raise questions aboutthe subsequent fate of such mononucleate progeny and thepotential participation of endogenous myofibers in the limbstump during regeneration.

MATERIALS AND METHODSCell Culture. Primary cultures of newt limb cells were

propagated as described (13). Myogenesis was induced inconfluent plates of limb cells by lowering the serum concen-tration from 10% to 0.5-1%. After 4 days, >90% ofcells fusedinto multinucleate myotubes that contained up to severalhundred nuclei. Cells were treated with trypsin and sievedthrough a 100-,m nylon mesh (CellMicroSieve; BioDesign,New York) to remove clumps, followed by passage througha 35-,um mesh to remove mononucleate cells. Myotubes wereretained by the mesh and were further enriched during a brief(0.5-1 hr) preplating period, after which most of the remain-ing mononucleate cells preferentially adhered to the sub-strate. The resulting population, which included approxi-mately equal numbers of myotubes and mononucleate cells,was replated at low density (<10 myotubes per mm2) formicroinjection.For [3H]thymidine labeling experiments, proliferating

mononucleate cultures were exposed to 1 mCi of [3H]thymi-dine per ml (1 Ci = 37 GBq) for 2 days prior to induction ofmyogenesis. Approximately 25% of the nuclei containedwithin myotubes were 3H labeled. Myotubes received mi-croinjections of rhodamine-dextran 4 days after inducingmyogenesis. For assessment of [3H]thymidine incorporationin culture, cells were fixed in 4% paraformaldehyde, washed,dehydrated, coated with photographic emulsion (1:1; IlfordK-5), and developed the next day.

Microinjection and Implantation. At 1-2 days after replat-ing, 10% rhodamine-conjugated lysinated dextran dissolvedin sterile water (Molecular Probes D-1817; Mr, 10,000) wasmicroinjected into visually identified myotubes with an Ep-pendorf semiautomated microinjection apparatus on a Zeissinverted microscope. At 1-2 days after microinjection, la-beled myotubes were counted, treated with trypsin, mixedwith 8000-10,000 unlabeled mononucleate cells as carrier,and pelleted by centrifugation in small-bore siliconized Pas-teur pipettes (0.5 mm; Billbate Glass, Daventry, U.K.)plugged with Sylgard 184 elastomer (Dow-Coming). Cellpellets were released from pipettes by scoring and breakingthe glass. Pellets were implanted 3-7 days after midfemoralamputations of adult newts (Notophthalmus viridescens)with the contralateral blastema as control. Wound epitheliawere released from underlying tissue using iridectomy scis-sors, forming "pockets" into which pellets were inserted;

*Present address: Department of Neurobiology, Duke UniversityMedical Center, Box 3209, Durham, NC 27710.tTo whom reprint requests should be addressed.

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wound epithelia were then reattached by using a cyanoacry-late ester adhesive (Histoacryl Blue). Overall cell loss duringimplantation was generally <10%.

In some instances, myotubes were labeled with the rho-damine-dextran tracer by microinjection as described butwere left in coculture with proliferating mononucleate blast-emal cells at high or low density. Labeled mononucleate cellswere never observed in these cultures for periods up to 6-8weeks, indicating (i) that under the conditions of cell culturededifferentiation did not occur and (ii) that the tracer couldnot be transferred from myotubes to mononucleate cells.

Histology. Blastemas were fixed by cardiac perfusion with24% paraformaldehyde, embedded, frozen, and seriallysectioned along the proximodistal axis at 50-100 ,um using aLeitz sledge microtome with freezing stage. Sections werecounterstained with Hoechst dye 33258 (bisbenzimide;Sigma) (1.25 Mg/ml) to visualize cell nuclei and mounted ongelatin-coated slides in 1% Dabco (1,4-diazabicyclo[2.2.2]-octane; Aldrich) in 90% (vol/vol) glycerol/10% phosphate-buffered saline, pH 8.6. For autoradiography, blastemaswere serially sectioned at 25 ,um, dehydrated, coated withphotographic emulsion (1:1; Ilford K-5), developed 1-2 dayslater, counterstained, and mounted similarly. Sections wereviewed with a Zeiss Axiophot photomicroscope equippedwith differential interference contrast optics and epifluores-cence using standard filter sets for Hoechst dye and rhoda-mine.

RESULTS AND DISCUSSIONLabeling and Implantation of Myotubes. Limb regeneration

in urodeles proceeds by rapid migration of epidermal cells

Myotube sieving

ImkrhTraicter

microinjection

I

Cell pelleting

if

* Inmplantation and glueing

1 week 2-3 weeks 4-6 weeks

FIG. 1. Schematic representation of an implantation experiment.Lineage tracer was injected into individual myotubes previouslyinduced in culture and purified; labeled myotubes were pelleted andimplanted beneath the wound epidermis. Limbs were allowed toregenerate, and blastemal tissue was collected after the three periodsshown.

FIG. 2. Labeling of cultured newt myotubes by injection of fluorescent lineage tracer. Myotube that received a microinjection of rhodaminelysinated dextran and was surrounded by mononucleate cells. (A) Phase-contrast microscopy. (B) Fluorescence optics. A myotube previouslyincubated in [3H]thymidine and microinjected was visualized by fluorescence double exposure to show rhodamine-labeled cytoplasm and blueHoechst-stained nuclei (C) or bright-field and fluorescence optics to show nuclei with silver grains (D). Arrows indicate nuclei in the myotube.(Bar = 200,m.)

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across the amputation plane to form the wound epithelium,followed by accumulation of mesenchymal blastemal cells inthe first 2 weeks (14). It is during this period that mesenchy-mal tissue may dedifferentiate and, hence, when labeledmyotubes were implanted. Myogenesis was induced in cul-tured newt limb cells by reduction of serum in the growthmedium (13). The myotubes that formed were differentiatedmuscle cells as judged by several criteria; namely, (i) theywere multinucleate, containing as many as several hundrednuclei within a single myotube; (ii) they lost expression oftheblastemal-specific antigen 22/18 (which is expressed by

FIG. 3. Examples of labeled mononucleate cells in sections ofregenerating limbs 9-10 days after implantation oflabeled myotubes.(A) Three mononucleate cells with rhodamine-labeled cytoplasm andHoechst-stained nuclei. (B) Mononucleate cell labeled with [3H]thy-midine and visualized by Hoechst staining after autoradiography todemonstrate the nucleus with silver grains. (C) Mononucleate cellshown in B labeled by fluorescence to show the rhodamine-labeledcytoplasm. Labeled mononucleate cells were generally well sepa-rated by excess numbers of unlabeled cells and could be identifiedunambiguously by distinct rhodamine-dextran labeling of the cyto-plasm surrounding single, Hoechst-stained nuclei (A). In contrast,multinucleate muscle fibers showed striking cytoplasmic labelingthroughout their lengths and the characteristic peripheral arrange-ment of Hoechst-stained nuclei (see Fig. 5B). (Bar = 50 ,um.)

mononucleate cells) and gained expression of the muscle-specific antigen 12/101 (9, 15); and (iii) they became con-tractile in response to mechanical stimulation.The myotube cultures were purified by sieving through

nylon mesh in order to remove mononucleate cells on thebasis of size (Fig. 1). Myotubes thus isolated were replated atlower density to facilitate their unambiguous identificationand subsequent microinjection of lineage tracer. The exper-iments reported here used rhodamine-conjugated lysinateddextran as a lineage tracer introduced through cytoplasmic

FIG. 4. Fluorescence photomontages of longitudinal sectionsthrough blastemas after implantation of labeled myotubes. (A) At 13days postimplantation, most cells are mononucleate and spreadthrough the developing limb, although there are some multinucleatecells (arrow) and also a labeled myotube (m) incorporated in themuscle mass. (B) At 24 days postimplantation, only a few labeledmononucleate cells are visible (open arrow), peripheral to thedeveloping cartilage, which occupies the central third of the regen-erate tissue. Note that the epidermis and bone (*) are autofluores-cent. Labeled cells were never detected in the blastemal mesen-chyme when animals received implantations containing cells that hadnot been injected with lineage tracer. (Bar = 500 Am.)

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microinjection (Fig. 2 A and B). Rhodamine-dextran is cellimpermeant and has been used extensively as a tracer for celllineage and developmental fate in a variety of organisms (see,for example, refs. 16-18). Rhodamine-dextran was not takenup by the cultured newt cells from the medium or transferredfrom labeled to nonlabeled cells even in very dense culture(see Materials and Methods). Only myotubes containing atleast 5 nuclei received microinjections; on average, labeledmyotubes contained 8-10 nuclei. For double-labeling exper-iments, proliferating mononucleate cells were exposed to[3H]thymidine prior to serum removal in order to labelmyotube nuclei (Fig. 2 C and D). Myotubes were pelleted andimplanted under the wound epithelium 3-7 days after hind-limb amputation (Fig. 1; see Materials and Methods).

Immediately before collecting and pelleting injected myo-tubes, cultures were checked under fluorescence microscopyfor the presence oflabeled mononucleate cells in the cultures.Of the 23 implantations of 50-100 myotubes described here,16 contained no detectable mononucleate cells, 5 containedone labeled mononucleate cell, and 2 contained four suchcells. Since we observed hundreds to thousands of stronglylabeled mononucleate cells in blastemas after implantation(see below), in no case was it possible that a significantnumber of these cells arose from the inclusion of labeledmononucleate cells in the implants.

Analysis of Blastemas. Blastemas that received implantsgave rise to normal regenerates, sometimes with a few daysdelay compared to unoperated contralateral limbs. A total of23 blastemas were prepared for serial sectioning during threeperiods after implantation: (i) 1 week (5-9 days), early in the

phase ofblastemal cell formation and proliferation (n = 8); (ii)2-3 weeks, in the phase of blastemal cell proliferation (n =10); (iii) 4-6 weeks, when differentiation, particularly ofcartilage, is becoming apparent (n = 5). As shown in Fig. 3,significant dedifferentiation was observed in blastemas ex-amined 1 week after implantation. Up to 100 mononucleatecells strongly labeled with the rhodamine-dextran tracer wereevident in these blastemas, distributed broadly around theregion of the implanted pellet, and extending through ap-proximately one-third of the blastema. These cells wereclearly mononucleate as evidenced by nuclear counterstain-ing (Fig. 3A) and could be double-labeled with [3H]thymidineas demonstrated after autoradiography (Fig. 3 B and C). Theoccurrence of double-labeled cells displaying both nuclearand cytoplasmic tracers demonstrates that the rhodamine-dextran tracer is transferred to cells arising directly from theoriginal population of labeled myotubes.By 2-3 weeks postimplantation, when blastemas were at

the midcone stage of regeneration, the dedifferentiation ofmyotubes was essentially complete. Hundreds to thousandsof labeled mononucleate cells, but relatively few multinucle-ate cells, were observed; labeled mononucleate cells wereseen dispersed throughout the blastema (Fig. 4A). The pro-portion of myotubes that underwent dedifferentiation is dif-ficult to assess from these observations because the numberof elapsed cell divisions is unknown. Nonetheless, assuminga cell cycle time of 2-3 days (19), a conservative estimateyields a lower limit of 10-15%. The actual proportion ofmyotubes that dedifferentiated could well have exceeded50% if the overall rate of division was slower or if cell death

FIG. 5. Muscle fiber and cartilage cells in sections of regenerating limbs after implantation of labeled myotubes. (A) Labeled muscle fiber9 days after implantation, showing striations under differential interference optics. (B) Rhodamine labeling of cytoplasm and Hoechst-stainednuclei at the periphery. (C) Differentiating cartilage shown under differential interference optics 26 days after implantation. (D) Same as C exceptunder fluorescence optics to show two adjacent rhodamine-labeled cells with Hoechst-stained nuclei. Cartilage was identified by its regular arrayof nuclei within the translucent cartilage matrix and by encasement of chondrocytes in lacunae. (Bar = 100 pm.)

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was significant. The numerous examples of strongly labeledmuscle fibers (Fig. 5 A and B) at this stage indicated thatfusion of labeled cells into existing muscle fibers oftenoccurred. Our experiments did not address whether labeledmuscle fibers arose from fusion with labeled myotubes fromthe original implant or from fusion with labeled mononucleatecells deriving from the labeled myotubes.At 4-6 weeks after implantation, cores of cartilage had

differentiated in the regenerates and digit formation wasobserved. Cartilage can be identified unambiguously in thesetissue sections by its regular array of nuclei, the translucentappearance of the cartilage matrix, and the encasement ofchondrocytes within lacunae. The staining intensity of la-beled cells was weaker at this stage, presumably from dilu-tion of the lineage tracer through their continued prolifera-tion. Nevertheless, our preliminary observations indicatedthat labeled mononucleate cells were widely distributed insuch regenerates, almost exclusively in undifferentiated mes-enchyme (Fig. 4B). Labeled cells were occasionally seenwithin cartilage. Interestingly, such cells were typically seenin clusters oftwo to four (Fig. 5 C and D), suggesting that theyderived from a single cell that had recently committed tochondrogenesis. These findings were consistent with thelimited occurrence of transdifferentiation from muscle tocartilage. Although morphological identification of cartilagecells in newt limb tissue is quite reliable, it will be necessaryto confirm these observations by use of molecular markers.These results demonstrate that implanted myotubes are

induced by the environment of the limb blastema to dedif-ferentiate into mononucleate cells, which then proliferate. Incontrast, we have not observed the dedifferentiation of newtmyotubes left in culture (see Materials and Methods), pro-viding strong evidence that extrinsic factors can alter thestability of the differentiated state. The eventual fate of themajority oflabeled mononucleate cells remains unclear, sincethe lineage tracer did not persist reliably beyond 4-6 weeks.Further experiments regarding the differentiation of thesecells using more persistent lineage tracers will be of great

interest, particularly in light of our preliminary observationsofthe limited occurrence oflabeled cells in cartilage, possiblyreflecting transdifferentiation. The availability of culturednewt myotubes offers the possibility of pursuing the cellularand molecular mechanisms of dedifferentiation in vitro.

We would like to thank P. Ferretti and A. Gann for helpfulcomments on the manuscript. D.C.L. was supported by a fellowshipfrom the Human Frontiers Science Program.

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