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Skin Regeneration in Adult Axolotls: A Blueprint for Scar- Free Healing in Vertebrates
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Title: Skin Regeneration in Adult Axolotls: A Blueprint for Scar- Free Healing in Vertebrates
Series Title: Seifert AW, Monaghan JR, Voss SR, Maden M (2012) Skin Regeneration in Adult Axolotls: A Blueprint for Scar-Free Healing in Vertebrates. PLoS ONE 7(4): e32875. doi:10.1371/journal.pone.0032875
Physical Description: Journal Article
Creator: Seifert, Ashley W.
Publisher: Public Library of Science (PLOS)
Place of Publication: internet
Publication Date: April 2, 2012
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Abstract: While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair.
Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Ashley Seifert.
Publication Status: Published
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Source Institution: University of Florida Institutional Repository
Holding Location: University of Florida
Rights Management: Applicable rights reserved.
System ID: IR00001311:00001

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SkinRegenerationinAdultAxolotls:ABlueprintforScarFreeHealinginVertebrates AshleyW.Seifert 1 ,JamesR.Monaghan 1 ,S.RandalVoss 2,3 ,MalcolmMaden 1 1 DepartmentofBiology,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmerica, 2 DepartmentofBiology,UniversityofKentucky,Lexington,Kentucky,United StatesofAmerica, 3 SpinalCordandBrainInjuryResearchCenter,UniversityofKentucky,Lexington,Kentucky,UnitedStatesofAmerica Abstract Whileconsiderableprogresshasbeenmadetowardsunderstandingthecomplexprocessesandpathwaysthatregulate humanwoundhealing,regenerativemedicinehasbeenunabletodeveloptherapiesthatcoaxthenaturalwound environmenttohealscar-free.Theinabilitytoinduceperfectskinregenerationstemspartlyfromourlimitedunderstanding ofhowscar-freehealingoccursinanaturalsetting.Herewehaveinvestigatedthewoundrepairprocessinadultaxolotls anddemonstratethattheyarecapableofperfectlyrepairingfullthicknessexcisionalwoundsmadeontheflank.Inthe contextofmammalianwoundrepair,ourfindingsrevealasubstantialreductioninhemostasis,reducedneutrophil infiltrationandarelativelylongdelayinproductionofnewextracellularmatrix(ECM)duringscar-freehealing.Additionally, wetestthehypothesisthatmetamorphosisleadstoscarringandinsteadshowthatterrestrialaxolotlsalsohealscar-free, albeitataslowerrate.AnalysisofnewlyformingdermalECMsuggeststhatlowlevelsoffibronectinandhighlevelsof tenascin-Cpromoteregenerationinlieuofscarring.Lastly,ageneticanalysisduringwoundhealingcomparingepidermis betweenaquaticandterrestrialaxolotlssuggeststhatmatrixmetalloproteinasesmayregulatethefibroticresponse.Our findingsoutlineablueprinttounderstandthecellularandmolecularmechanismscoordinatingscar-freehealingthatwillbe usefultowardselucidatingnewregenerativetherapiestargetingfibrosisandwoundrepair. Citation: SeifertAW,MonaghanJR,VossSR,MadenM(2012)SkinRegenerationinAdultAxolotls:ABlueprintforScar-FreeHealinginVertebrates.PLoSONE7(4): e32875.doi:10.1371/journal.pone.0032875 Editor: ZhongjunZhou,TheUniversityofHongKong,HongKong Received September15,2011; Accepted February3,2012; Published April2,2012 Copyright: 2012Seifertetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: TheAmbystomaAffymetrixGenechipsweredesignedfromatranscriptassemblyfundedbyNIH-NCRR(R24-RR016344).Mostofthetranscriptswere generatedundertheNIH-NCRRgrantandanArmyResearchOfficegrant(MURI:W911NF-09-1-0305)toKenMuneoka,DavidGardiner,andSRV.Additional transcriptswereprovidedbyEllyTanakathroughfundingfromtheDFGCenterforRegenerativeTherapies,andBerndFritschthroughfundingfromNIHNIDCD (R01-DC005590-07S1).TheaxolotlswereobtainedfromtheAmbystomaGeneticStockCenterattheUniversityofKentucky,whichisfundedbytheNation al ScienceFoundation(DBI-0951484).ThisstudywassupportedbyNIH5RC2NS069480toMMandAWSisfundedbyNIH-NIDDK5T32DK074367.Publicationofthi s articlewasfundedinpartbytheUniversityofFloridaOpen-AccessPublishingFund.Thefundershadnoroleinstudydesign,datacollectionandanaly sis,decision topublish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:seifert@ufl.edu Introduction Amongitsmanyfunctions,theskinisprimarilyresponsiblefor maintainingthestructuralandphysiologicalbarrierbetweenan organism'sinternalandexternalenvironment.Asthefirstlineof defenseagainstexternalinsulttheskinisinjuredmorefrequently thananyothertissueandresultingdamage,whilerepairable,leads topermanentscarringinmammals[1].Atleast100millionpeople inthedevelopedworldacquirescarseachyearinresponseto traumaandsurgeryandtheresultisaspectrumofpathologies fromthinlinesurgicalscarstohypertrophicandchronicnonhealingwounds[2,3,4].Addingtothismedicalburden,burn injuries,whichoftenelicitanover-exuberantfibroticresponse, resultinhypertrophicscarringwithtreatmentcostsintheUS aloneaccountingfor $ 4billionannually[5].Whilenotascomplex asregeneratingahumandigitorlimb,theabilitytodevelop regenerativestrategiesthatleadtoscar-freehealinginadultskin remaintantalizinglyoutofreach.Understandinghowtocoaxthe naturalwoundrepairprocesstowardsaregenerativeoutcome remainsthegrailofwoundhealingresearch. Ourknowledgeofthemolecularandcellulareventsduring mammaliantissuerepairisextensive(seerefs[1,6,7,8])andyet, evenwithsuchbroadunderstandingofthewoundrepairprocess, regenerativemedicinehasfailedtodeveloptherapiesthatcan perfectlyregenerateskin.Thisstemspartlyfromthedynamic reciprocityofcellularinteractionsandsignalingpathwaysand partlyfromalackofappropriatemodelstoobservethese interactionsinaregenerativeenvironment[9].Whilewound repairinfetalmammals[10,11,12,13,14]andmarsupials[15]has providedinsightintothecellularandmolecularregulationofscarfreehealing,comparisonsofwoundrepairbetweenfetalmammals andadultshaslimitations,bothbiologicalandpractical[16].The developingfetus,atthetimewhenithealsscar-free,hasan immatureendocrinesystem,isimmuno-incompetent,iscontained inamoiststerileenvironment,anditscellsareinastateofchronic hypoxia[16].Adultskinismorecompletelydifferentiatedand adultwoundsareopentodesiccationandinfection,twofactors thatseriouslycomplicatewoundrepair.Otherpromisingmodels ofscar-freehealing,suchastheMRLmouse,whichsharethe abilitytoregenerateearpuncheswithrabbits,hares,pikas,cows, pigsandcats[17,18,19]hasprovenlessthanperfectwhen challengedtohealexcisionalskinwounds[20,21]castingdoubton thespecialregenerativepowersofthisinbredmousemodel. Comparedtoothervertebrates,urodelespossesstheamazing capacitytoregeneratetheirlimbs,hearts,lenses,spinalcords,tails, internalorgansandjoints.Observationsfromstudiesexamining PLoSONE|www.plosone.org1April2012|Volume7|Issue4|e32875

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limbregenerationhavebeenextrapolatedtotheskin,butdirect comparisonstotheprocessesofcutaneouswoundrepairhave rarelybeenmade[22].Studiesexamininglimbwoundshave yieldedinsightintotheprocessofre-epithelialization[23,24,25,26] andregenerationofthebasementmembrane[27,28],butthe dynamicsofdermalregenerationhaveremainedobscure.Direct studyofwoundrepairinurodeleskinoutsideofregenerationfields likethelimbandtail,however,hasnotbeenundertaken. Giventheirseeminglyabsolutepowersofregeneration,a recurringquestionhasbeenwhetherwoundsmadeoutsideof regeneratingstructures(e.g.limbsandtails)inadulturodelesare capableofscar-freehealingor,likeadultanurans,healwithascar [29].Inthisstudyweexaminedfullthicknessexcisional(FTE) woundhealingofdorsalbackskininadultaxolotls.Usingan establishedmammalianexcisionalwoundmodeltodirectly characterizecutaneouswoundhealinginadultaxolotls,we examinedhemostasis,inflammation,newtissueformationand remodelingprocesses.Additionally,weinducedmetamorphosisin adultaxolotlstotestthehypothesisthatlossoflarvalskin charactersandtransitiontoaterrestrialformresultsinfibrotic scarringfollowingFTEflankwounds.Herewedemonstratethat bothaquaticandterrestrialaxolotlsarecapableofperfect,scarfreeskinregeneration.Wediscussthesefindingsinthecontextof mammalianwoundrepairandpresentablueprintforinvestigating thecellularandmolecularmechanismsthatregulatescar-freeskin healinginadultvertebrates. Results FullThicknessExcisionalFlankWoundsarePerfectly RegeneratedinAdultAxolotls Adultaxolotls( Ambystomamexicanum )areaquaticandpaedomorphic,exhibitingseveraljuvenilefeaturesasadults(e.g. retentionofleydigcells,pseudo-stratifiedepidermis,externalgills). Althoughthedermisofadultaxolotlskinistypicalforamphibians, theepidermisispseudo-stratifiedandlacksawell-definedstratum corneum(Figure1A,B).Abovethestratumgerminativum, epithelialcellsareinterspersedwithleydigcells(specializedcells containinghighlygranulatedcytoplasm)thatarecharacteristicof larvalamphibianskin(Figure1Band[30,31]).Thedermis containsepidermally-derivedmucousandgranularglandsthatare embeddedwithinthestratumspongiosum,aloosenetworkofthin collagenfibersandfibroblaststhatliesabovethestratum compactum(Figure1A,BandFigureS1A,B).Thestratum compactumformsathickenedsheetofcompressedcollagenfibers thatsitsatophypodermisandseparatestheskinfromthe underlyingmuscle(Figure1A). Ithasbeenreportedthatwoundsmadeoutsideofregeneration fields(i.e.limbs,tail,head)healwithascar[32]andtodate,this controversyremainsunresolved.Todirectlytestthehypothesis thataxolotlscanperfectlyregenerate(i.e.healscar-free)full thicknessexcisional(FTE)wounds,wemadecircular4mmFTE woundsthroughthedermisintothedorsalflankmuscle(n=12) andobservedthewoundhealingprocessover180days(Figure1CH,Figure2,FiguresS2).Inresponsetoinjurybloodflowedinto thewoundbed,clotted,butdidnotformascab(Figure2). Epithelialcellsfromthewoundmarginsmigratedacrossthe underlyingmuscleandaccumulatedplasmaandcompletelyreepithelializedthewoundwithin24hrs(Figure1C,greenarrows indicateplasma).Followingre-epithelializationnumerousblood cells(leukocytesanderythrocytes)wereapparentinthewoundbed andtheneoepidermisremainedincloseproximitywiththe underlyingmuscleandplasmaoverthenext710days (Figure1D).Fourteendayspostinjuryweobserveddermal fibroblastsinthewoundbedandMasson'sTrichromestaining revealednewlydepositedextracellularmatrix(ECM)(Figure1E). Twenty-onedayspostinjurymusclefiberscontinuedtofragment liberatingmono-nucleatecellsintothesurroundingtissueanda robustECMformedbetweentheepidermisandmuscle(Figure1E, F).Forty-sevendayspost-injurynewepidermalorganswere presentintheregeneratedstratumspongiosum(Figure1Gand FigureS2A).Thestratumcompactum,however,hadnot completelyregeneratedandthiswasclearlyevidentatitsmargins (Figure1GandFigureS2B).Althoughthetimingtoregeneration variedamongindividuals,fullthicknessskin,includingepidermal organsandunderlyingmuscle,wascompletelyregeneratedby 80days(Figure1HandFigure2).Maturationanddevelopmentof glandsandthestratumcompactumcontinuedoverthenext 100days(FigureS2C). NewECMDepositionCorrespondswithFormationofthe LaminaDensa Previousworkinregeneratingnewtlimbssuggestedthat reformationofthebasementmembrane(BM)facilitatesdermal regenerationanditsdelayedformationpermitsblastemaformation[28].WefollowedBMregenerationafterre-epithelialization andaskedwhetheritoccurredpriortotheonsetofdermal regenerationinexcisionalflankwounds.InuninjuredskintheBM isvisibleasathickfibrousbandseparatingepidermisfromdermis andiscontinuousexceptwheremucousglandsinterjectintothe epidermis(Figure3A).Followingre-epithelializationhistological stainingrevealedathin,immaturestructurebeneaththenew epidermis(Figure3A).TheBMcontinuedtomatureandwas completelyregeneratedatleast47daysafterwounding(Figure3A; yellowarrowsD47).Interestingly,completeregenerationofthe BMcorrespondedtoregenerationofthedermis(exceptfor stratumspongiosum)(Figure3AandFigure1G). InordertotestifassemblyandmaturationoftheBM correspondedtotheonsetofECMdepositionweanalyzed formationofthelaminalucidaandlaminadensausingantibodies thatrecognizelaminin(laminalucida)andcollagentypeIV (laminadensa)(Figure3B).Bothproteinsweredetectableinthe BMofuninjuredskin,andsurroundingglandsandmusclefibers (Figure3B).Followingre-epithelialization,basalepidermalcells werenegativeforbothlamininandcollagenIV(Figure3B;white arrows).Wedetectedstrongandcontinuouslamininstaining beneaththeepidermis7dayspostinjuryindicatinglaminalucida reformation(Figure3B).CollagenIVwasnotdetectedcontinuouslybeneaththeepidermisuntilD14(correspondingtotheonset ofECMdeposition)(Figure3B). Inadditiontoproteinlocalizationwetrackedepidermalgene expressionoftheBMcomponents collagentypeIV and lamininalpha 1( Lama1 ), lamininbeta 1( Lamb1 ), laminingamma 1( Lamc1 ),which togetherformtheproteinlaminin 111(TableS1).Expressionofall threelaminin-111subunitswasunchangedduringre-epithelialization(TableS1).Expressionofboth alpha-1 and beta-1 componentsoflaminin-111increased5.6-foldand4.1-fold respectivelybetweenD1andD3,andcontinuedincreasingat D7postinjury(TableS1).Intheskinbasementmembraneof humansandmice,twohetero-trimericcollagentypeIVmolecules consistingofalpha1(IV) 2 /alpha2(IV)andalpha5(IV) 2 /alpha6(IV) chainsexist[33].Weexaminedexpressionofeachofthesealpha chainsfollowinginjuryandfoundthatexpressionof alpha1 and alpha2 chainsweredownregulatedfollowinginjury,remained belowbaselinelevelsuntilD3,afterwhichtheyreturnedto baselinelevelsatD7postinjury(TableS1).Whereasexpressionof the alpha6 chainremainedunchanged,the alpha5 chainwas upregulated4.9-foldatD1andsustainedthroughD7(TableS1). Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org2April2012|Volume7|Issue4|e32875

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Takentogether,theseresultsdemonstratethatregenerationofthe BMproceedsthroughformationofthelaminalucidafollowedby productionofthelaminadensaandthatcompletelaminadensa formationcorrespondstotheonsetofECMproductioninthe woundbed. MetamorphicAxolotlsExhibitaDelayinSkin RegenerationComparedtoPaedomorphicAxolotls Whileadultmammalsareincapableofregeneratingfull thicknessskinwounds,fetalmammalsexhibitscarlesshealingof similartypewounds[12].Similarly,whilepre-metamorphic anuranshealscar-free,post-metamorphicanuranshavebeen documentedtohealflankwoundsthroughscarformation[29]. Adultaxolotlsretainseverallarvalskinfeatures(e.g.leydigcells, pseudo-stratifiedepithelium),thusweaskedifthesecharacteristics facilitatetheirabilitytohealwoundsscar-free.Totestthis hypothesisweexploitedthefactthatnormallyaquaticaxolotls retaintheabilitytoundergometamorphosistoaterrestrialform throughadministrationofthyroxineandweinducedmetamorphosisinadultaxolotls(controllingforageandsizewithsibling paedomorphs).Comparinguninjuredepidermisbetweenboth formswenotedtwomajordifferences;first,granularglandsthat occupiedrelativelylittlespaceinthepaedomorphdermiswere greatlyenlargedandoccupiedmostofthestratumspongiosum whilemucousglandsappearedsimilarinformbetweenmorphs (Figure4AandFigureS3A).Second,theepidermisnolonger containedleydigcellsandhadtransitionedtoacompletely stratifiedepitheliumexhibitingawell-definedstratum germinativum,stratumspinosum,stratumgranulosumand stratumcorneum(Figure4B). Wenextexaminedwoundrepairinmetamorphsfollowing 4mmfullthicknessexcisionalwoundsover180days(Figure4C-H Figure1.Scar-freehealingoffullthicknessexcisionalwoundsinadultaxolotls. A)Masson'strichromestainingofuninjureddorsalflank skininaxolotlsshowingepidermis(E),dermis(D),hypodermis(H)andunderlyingmuscle(M).B)Magnifiedimageofepidermisanddermis.The epidermisispseudo-stratifiedandcontainsepithelialcellsandleydigcells(yellowarrows)whilethedermisisdividedintothestratumspongios um (containingglandsanddermalfibroblasts)atopthedenselycompactedECMofthestratumcompactum.C-H)Scar-freehealingover80dayperiod followingfullthicknessexcisionalwounding.C)Onedaypostinjury(dpi)thewoundbediscompletelyre-epithelialized.Somebloodplasmahas accumulatedbeneaththeneoepidermis(greenarrows)(woundmargin=WM).D)Sevendpithereislittleevidenceofafibrinclotbetweenthe epidermisandunderlyingmuscleandnonewECMhasbeendeposited.Greenarrowsdepictresidualbloodplasma.E)Fourteendpifibroblastsare visiblebeneaththeepidermiswherenewECMisdeposited(bluestaining)andmusclecellsarefragmentingintoindividualmyoblasts.F)Twenty-one dpiathickbandoftransitionalmatrixisvisiblebeneaththeepidermis.Collagenisvisiblewithintheregeneratingmuscle.G)Forty-sevendpithe underlyingmusclehascompletelyregeneratedandisdevoidofcollagen.Skinglands(yellowarrow)haveregeneratedanddescendedfromthe epidermis.Thedermalstratumspongiosumhasreformedandthestratumcompactumiscoalescing.H)Eightydpiallskinlayershaveregenerated. Scalebars=100 m m. doi:10.1371/journal.pone.0032875.g001 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org3April2012|Volume7|Issue4|e32875

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andFigureS3B-E).Twenty-fourhourspostinjuryre-epithelializationwasnotcompleteandtheleadingedgeofmigrating epidermalcellswasvisiblebothmacroscopicallyandinsection (Figure4C).Re-epithelializationwascompleteby72hourspost injuryandtheepidermishadre-stratifiedtoestablishapical/basal polarityalongtheregeneratingbasementmembrane(Figure4D). Sevendayspostinjurythewoundbedcontainedplasmabeneath theepidermisandwithinthefragmentedmuscle,alongwithlarge numbersoferythrocytesandleukocytes(Figure4D).ECM depositionbeganasithadinthepaedomorphapproximately 1014dayspostinjuryandwenotedthatitappearedtoextend deepintothemuscle(Figure4E).Twenty-onedayspostinjurythe ECMwasdensewithinthewoundbed,newvasculaturewas presentinboththeepidermisandECM,musclefiberscontinued tofragmentandaggregationsofcellsappearedintheepidermis suggestingglandswerebeginningtoregenerate(Figure4Fand FigureS3B). Completedermalregenerationwasdelayedinmetamorphs (compareFigure1GandFigure4G).Whileepidermalorgans regeneratedinbothformsafter40days,thewoundbedand underlyingmusclestillcontaineddenselycompactedextracellular matrixinmetamorphs(Figure4GandFigureS3C).After80 + days thestratumspongiosumhadregeneratedbutthestratum compactumremainedincomplete(Figure4H).After120days, thewoundsiteresembledan80-dayregeneratingwoundin paedomorphsandafewcollagendepositsstillpersistedinthe underlyingmuscle(FigureS3D).Fibrosiswasnotresolveduntilat least148daysandwhilemucousglandsregeneratedtopre-wound size,granularglandsremainedsmallevenafter148days(Figure S3E).Takentogetherthesefindingssuggestthatflankskininadult metamorphicaxolotlscancompletelyregeneratefollowingFTE wounding,butthetimerequiredtoregenerateboththestratum compactumandmaturegranularglandsislengthenedcompared topaedomorphs. TheRateofRe-epithelializationisSlowerinMetamorphs Followinginjury,re-epithelializationofthewoundbedis necessarytore-establishepidermalintegrity.Invariousrodent species,untreatedFTEwoundsre-epithelializebetween3and 7days(personalobser.).Incontrast,aquaticaxolotlsre-epithelialize4mmflankwoundsin 18hrs(Figure1CandFigureS4).We comparedtherateofre-epithelializationbetweenpaedomorphs andmetamorphstotestwhetherthecompletelystratified epidermisofterrestrialaxolotlsaffecteditsabilitytocoverthe woundbed.Usinganantibodytoawide-spectrumofcytokeratins welabeledtheepidermis24and72hrsafterinjury(Figure5). Comparedtothecompletelyre-epithelializedwoundbedof paedomorphs,woundedgeepithelialcellsinmetamorphshadjust begunmigrating24hrsafterinjury(Figure5A,B).Metamorphreepithelializationwascompleteby72hrs(Figure5C).Examination ofthewoundepidermisinpaedomorphsshowedleydigcells presentinthenewepidermis(Figure5D).Theleadingedgeof metamorphepidermisappearedpseudo-stratified(reminiscentof paedomorphepidermiswithoutleydigcells)andmovedasasheet ofcellswithasinglecellattheleadingedge(Figure5E).Following re-epithelialization,thewoundepidermisre-establishedastratified epithelialmorphology(Figure5F).Inconjunctionwiththisdelay inre-epithelializationweobservedaconcomitantdelayin appearanceoftheBMcomponentslamininandcollagentype IVreflectingthedependencyofBMreassemblyonthetimingof completere-epithelialization(datanotshown).Weexaminedthe expressionofthesamemolecularcomponentsoflamininand collagentypeIVaswedidforpaedomorphsanddidnotdetecta significantincreaseinexpressionpriortoD7(TableS1).These resultsshowthatstratifiedepidermisinmetamorphsrequiresan extendedactivationperiod(relativetopaedomorphs)before migrationbeginsandthisdelaycontributestoaslowerrateof re-epithelialization. Figure2.Scar-freehealinginadultaxolotls. Regenerationofdorsalflankskinfollowing4mmfullthicknessbiopsypunchwounding.White circlesdepictareaoforiginalinjury.IndividualpigmentcellsarevisibleatD14intheoverlyingepithelium.Regenerationandscar-freehealing atD89. ContractionisevidentatthewoundedgesafterD14.Scalebars=2.0mm. doi:10.1371/journal.pone.0032875.g002 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org4April2012|Volume7|Issue4|e32875

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MigratingKeratinocytesandNeoepidermisExpressHigh MMPLevels Epidermalexpressionandenzymaticactivityofserineproteinasesandmatrixmetalloproteinaseshasbeenshowntoaffect keratinocytemigrationandECMdegradationduringtheearly stagesofmammalianwoundhealing[1].Inordertobegin addressingtheroletheseenzymesplayduringskinregenerationin axolotlsweanalyzedtheexpressionoftheserineproteinase plasminogentissueactivator ( PLAT ),matrixmetalloproteinase( MMP ) familymembersandthe tissueinhibitorsofmetalloproteinases ( TIMP1 4 )inmigratingandneoepidermisfollowingexcisionalwoundingat D1,D3andD7inpaedomorphsandmetamorphs(TableS1). PLAT functionstoconvertplasminogentoplasminandconsistent withtheabsenceofafibrinclotwasnotexpressedeitherin uninjuredaxolotlskinorfollowinginjuryintheepidermis(Table S1). Duringskinregenerationinaxolotlswefoundastrong MMP responsetoinjuryinbothmigratingkeratinocytesandneoepidermisinpaedomorphsandmetamorphs(TableS1).Forall MMPs demonstratingsignificantchangesingeneexpression followinginjury,theexpressionkineticsgenerallyfollowedtwo patterns;(1)astrongupregulationatD1followedbyanequally strongdecreaseatD3andacontinueddecreaseorlevelingoffat D7and(2)astrongupregulationatD1whichremainedhigher thanbaselineatD3andthenwasdownregulatedatD7(Figure6). Thecollagenases MMP1 and axCOL (anamphibianspecific collagenaseorthologoustonewt nCOL )followedthefirstpattern with MMP1 exhibitingaparticularlystrongresponse,increasing 425-fold(paedomorphs)and502-fold(metamorphs)atD1,while remaining88-fold(paedomorphs)and61-foldelevated(metamorphs)atD7(TableS1).Thestromelysin MMP3/10a exhibited asimilarexpressionprofileasthecollegenasesexceptD7levels returnednearbaseline(Figure6). MMP3/10b levels,while exhibitingstrongupregulationatD1,exhibitedaslowdecreasing rateofexpression(pattern2)comparedto MMP3/10a and remainedelevatedatD7(Figure6).While MMP19 and MMP28 followedexpressionpattern1forpaedomorphs,expressionofboth genesremainedhighinmetamorphsmirroringthelagtimeinthe metamorphicre-epithelializationrate(Figure6).Thegelatinase MMP9 exhibitedpattern1expressionkineticsandresponded2foldgreaterinmetamorphs. MMP2 wastheoneexceptiontothe generallyobservedpatterns. MMP2 waseffectivelyturnedoffat D1,remainedoffatD3andthenwasupregulatedinboth paedomorphsandmetamorphs(Figure6).Giventhestrong Figure3.LaminalucidaandlaminadensaregeneratebeforenewECMdeposition. A)Histologicalexaminationofbasementmembrane (BM)regenerationinaxolotls.TheuninjuredBMisvisibleasathickblue-stainedfibrousband(yellowarrows).AnimmatureBMhasbeguntoreform (yellowarrowD1)afterre-epithelializationandisvisibleatthewoundmargin(WM)incontrasttotheuninjuredBM.TheregeneratedBMisvisibleat D47.YellowarrowsatD7andD21indicatereformingBM.B)Examinationoflaminalucida(laminin)andlaminadensa(collagentypeIV)during basementmembraneregeneration.TheuninjuredBMispositiveforlamininandcollagentypeIV(yellowarrows)asarethebasementmembranes surroundingglandsandmusclefibers.Followingre-epithelializationthebasallaminaoftheepidermisisnegativeforlamininandcollagentypeIV (whitearrows)andthisisclearlyevidentatthewoundmargin(WM).SevendayspostinjurytheBMstainsstronglyforlamininindicatingreformation ofthelaminalucida,whilestainingforcollagentypeIVispunctuated.ThelaminadensaisregeneratedbyD14basedoncontinuouscollagentypeIV stainingandpersistsduringdermalregeneration. doi:10.1371/journal.pone.0032875.g003 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org5April2012|Volume7|Issue4|e32875

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responsetoinjuryreflectedin MMP levels,wealsoanalyzed expressionlevelsforthe tissueinhibitorsofMMPs ( TIMPs )(TableS1). Only TIMP1 changedsignificantly,withpaedomorphsexhibiting pattern1expressionandmetamorphsexhibitingpattern2. Interestingly,axolotlsappeartohavetwocopiesof TIMP1 based onsequenceanalysisandthissecondcopydidnotchangeineither morphfollowinginjury(datanotshown).Previousexperiments duringnewtlimbregenerationhaveshownthat MMP transcriptioncorrelateswellwiththeirenzymaticactivity[34]andthehigh sequencesimilaritybetweennewtandaxolotl MMP sequences stronglysupportsMMPorthology.Weconductedgelatin zymographyduringpaedomorphictailregenerationandfound thattranscriptionandactivityofMMP9werewellcorrelated,thus supportingthisassociationinaxolotl(FigureS5A-C).This confirmsrecentworkduringaxolotllimbregenerationdemonstratingtheactivityofaxolotlMMP[35].Takentogetherthese resultssuggestMMPsmayplayakeyroleduringkeratinocyte migrationandregeneration,andsuggestthatsustainedactivity maycontrolthetimingofECMdeposition. MetamorphicAxolotlsExhibitIncreasedLeukocyte Infiltration Duringwoundrepair,adultmammalsexhibitastereotypical inflammatoryresponsecharacterizedbyinfiltrationandsubsequentremovalofleukocytes[36].Thetrade-offbetween regenerationandfibrosishasbeenpostulatedtoresultfroma weakinflammatoryresponsewithgreaterinflammationcontributingtoincreasedfibrosis[37].Inordertocharacterizethe inflammatoryresponseduringskinregeneration,weassayedthe woundbedforL-plastinpositivecells,apan-leukocyticmarker conservedinvertebrates.Tocontrolforindividualvariationwe harvestedwoundtissuefromthesameanimal(4woundsper animal)atD0,D1,D3,D7andD14frompaedomorphs(n=4) Figure4.Metamorphicaxolotlskinhealsscar-freebutslowercomparedtopaedomorphs. AB)Morphologyofuninjuredaxolotlskin aftermetamorphosis.A)Masson'strichromestainingshowingepidermis(E),dermis(D)containingenlargedgranularglandsinstratumspongiosum atopstratumcompactum,hypodermis(H)andmuscle(M).B)Highmagnificationofepidermisshowingstratifiedepithelium.Leydigcellshave disappearedandtheepidermisnowcontainsawell-definedstratumspinosum,granulosumandcorneum.C-H)WoundhealingfollowingFTE woundsover82-dayperiod.C)Onedaypostinjury(dpi)epithelialcellshavebeguntomigratebutthewoundisnotre-epithelialized.Erythrocytes arevisibleinthewoundbedandbetweenmusclefibersundergoinghistolysis.D)Re-epithelializationiscomplete3dpiandat7dpicoagulated plasma(greenarrows),erythrocytesandleukocytesarevisibleinthewoundbed.E)Fourteendpifibroblastsarevisibleinthewoundbedandnew ECMisdeposited(bluestaining).Thewoundmargin(WM)isstillclearlyvisible.F)Thewoundbed21dpiisrichinECM.ThisECMextendsdeepinto theunderlyingmusclefiberswhicharefragmentingintomyoblasts.G).Regeneratingglands(yellowarrows)arepresentinthedermis42dpiandthe stratumspongiosumisbeginningtodevelop.Theunderlyingmusclecontinuestoremaindamagedwithdeeppocketsofcollagenpersisting beneaththewound.H)Eightydpithedermisispartiallyregeneratedbutthestratumcompactumhasnotcoalesced.Somecollagenstillpersistsdeep inthemuscleandbothmucousandgranularglandshaveregenerated(yellowarrows).Scalebars=100 m m. doi:10.1371/journal.pone.0032875.g004 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org6April2012|Volume7|Issue4|e32875

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andmetamorphs(n=4).ExaminingthenumberofL-plastin positivecellswefoundbothmorphsexhibitedarobustinfiltration ofleukocytesintothewoundbed(Figure7AandFigureS6A).We useda2-wayANOVAtoexaminetheeffectsofmorph (metamorphvspaedomorph)anddaypostinjuryontotal leukocytenumbers(withindividualasarandomeffecttoallow forrepeatedsampling).Thisanalysisyieldedasmallp-valuefor theeffectofmorph(F=3.05,p=0.095).Anexaminationofeach individualovertimeshowedthatonepaedomorphexhibitedmuch higherlevelsofL-plastinpositivecells(suggestingchronicstressor sickness)comparedwiththeotherthreeindividuals(FigureS6B, C;paed4).Thereforeweanalyzedthedatawithoutthisindividual andfoundthatmetamorphshadasignificantlyhighernumberof leukocytesfollowinginjury(F=5.32,p=0.032)(Figure7A).This differencewasmostpronouncedbetweenD1andD3(Figure7A) whenre-epithelializationwascompleteinpaedomorphsbutnotin metamorphs. Inordertofurtherquantifytheinflammatoryresponsewe examinedneutrophilinfiltrationinpaedomorphsandmetamorphs.Usingapolyclonalantibodytomyloperoxidase(MPO) wecountedallneutrophilsin5 m msectionsofthewoundbed betweentheepidermisandunderlyingmusclefollowinginjury (Figure7B).Neutrophilswerepresentinlownumbersfollowing injury(comparedtomammals)andwerenotsignificantly differentbetweenpaedomorphs(n=8)andmetamorphs(n=8) (F=2.50,p=0.136).ExaminingMPOpositivecellsacrossall axolotls(n=16)theeffectofdayspostinjuryapproached significance(F=3.04,p=0.058)andpost-hoccomparisons Figure5.Therateofre-epithelializationisslowerinmetamorphscomparedtopaedomorphs. Epidermiswaslabeledwithapancytokeratinantibody.A)Thewoundbediscompletelyre-epithelialized24hrsafterinjuryinpaedomorphs.Thewoundmargins(WM)arevisible wherethestratumcompactumisdisrupted.B,C)Woundedgekeratinocyteshavejustbegunmigrating24hrsdpiandre-epithelializationis completeby72hrsdpiinmetamorphs.D)Leydigcellsarepresentinthepaedomorphneoepidermis.E)Theleadingedgeofmigratingmetamorph keratinocytes.Theepidermalcellsappeartomoveasagroupofcellswithonecellattheleadingedge.F)Afterre-epithelializationiscompletein metamorphstheepidermisbecomesre-stratified. doi:10.1371/journal.pone.0032875.g005 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org7April2012|Volume7|Issue4|e32875

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revealedasignificantdecreaseinneutrophillevelsatD7 (t=2.014,p=0.0226). Inmammalianwounds,neutrophilsrepresentthemajorityof leukocytespresentduringtheearlyinflammatoryresponseand compriseapproximately59%ofcirculatingleukocytes[38].Our datasuggestedthatneutrophilscomprisedarelativelysmall fractionofinfiltratingleukocytesinaxolotlsafterinjury,andwe askedifthiswasdueinparttolowlevelsincirculation.Usinga modifiedWright'sstaintoidentifyleukocytesandSudanBlackto accuratelyidentifytheneutrophilpopulation,wecalculated leukocyteprofiles(percentageoftotalleukocytes)forboth paedomorphs(n=5)andmetamorphs(n=5)(Figure7Cand TableS2).Wefoundthatleukocytescompriseapproximately4% ofcirculatingbloodcellswithneutrophilsaccountingfor 21% oftheleukocytepopulationinbothforms(TableS2).Comparing paedomorphsandmetamorphswefoundsignificantlyhigher eosinophillevelsinpaedomorphs(t=2.49,p=0.043)and significantlyhigherlymphocytelevelsinmetamorphs(t=3.96, p=0.004).Takentogether,theseresultsshowthataxolotls displayaninfluxofleukocytesfollowingfullthicknessexcisional woundingdemonstratinganinflammatoryresponsetowounding. However,relativetomammals,neutrophilscompriseasmaller fractionofinflammatorycellsatthewoundsite,andthis correlateswiththeirrelativelylowerrepresentationincirculating blood. Figure6. Matrixmetalloproteinase ( MMP )expressionduringre-epithelializationandnewtissueformation. Expressionvalues(y-axis) reflectabsoluteexpressionvaluesfromAffymetrixaxolotlgenechips(seeTableS1forexactvalues).Errorbarsrepresentstandarderror.Bluelin es representpaedomorphsandredlinesrepresentmetamorphs.Expressionkineticsforselected MMPs generallyfollowedtwopatterns;(1)astrong upregulationatD1followedbyanequallystrongdecreaseatD3andacontinueddecreaseorlevelingoffatD7[ MMP3/10a,MMP9 ]and(2)astrong upregulationatD1whichremainedhighatD3andthendownregulatedatD7[ MMP3/10b,MMP19 ].For MMP28 thekineticsfollowedpattern1for paedomorphsandpattern2formetamorphssuggestingthat MMP28 expressionwashighlyconnectedtore-epithelialization.Theexpressionprofile for MMP2 wasuniqueinthatiswasdownregulatedfollowinginjuryandwasupregulatedafterre-epithelializationwascomplete. doi:10.1371/journal.pone.0032875.g006 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org8April2012|Volume7|Issue4|e32875

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PercentWoundClosureMimicsContractionRatesDuring HealingofHumanSkinWounds Woundcontractionisanimportantcomponentoftherepair processandisanimportantmetricwhencomparingwoundrepair acrosstaxa[39].Inhumansandpigs,contractionofcircular woundsaccountsforlessthan50%ofwoundclosure,whilein rodentscontractionaccountsfor 90%ofrepair[39].Following excisionalwounding,thewoundareaincreasedslightlyin metamorphswhileremainingconstantinpaedomorphsandafter 24hrsthewoundsinbothgroupscontractedatapproximatelythe samerate(Figure8A).Quantifyingtherateofwoundcontraction betweenpaedomorphsandmetamorphs,wefoundthatpercent contractionwassignificantlygreaterinpaedomorphsandresulted inagreaterdegreeofwoundclosure(paedomorphs=67% 6 4.4% andmetamorphs=37.9% 6 9%)(Figure8B).Infact,ouranalysis showsthatmetamorphicaxolotlscontracttheirwoundsto approximatelythesamedegreeashumanskinafterwounding. Priortowoundcontraction,somedermalfibroblastsinthe granulationtissueandatthewoundmarginsbegintoexpress smoothmuscleactinandareresponsibleforgeneratingcontractile forcesthatpullthewoundmarginstogether[40,41].Wealso examinedlocalizationofalpha-smoothmuscleactin(alpha-SMA) (amarkerofmyofibroblasts)inpaedomorphsandcomparedits distributiontomiceatsimilartimepointsafterwounding(Fig.8C). Approximately10daysafterinjury,miceshowedahighnumber ofalpha-SMApositivecellsatthewoundmarginswhenthe woundareabeginstorapidlycontract(Figure8C;whitecircled area).Incontrast,wedidnotdetectmanyalpha-SMApositive cellsinaxolotlsatthewoundmarginsorinthewoundbedatD10, althoughthebasementmembranebeneaththewoundepidermis Figure7.Higherinitialleukocytelevelsarepresentinterrestrialaxolotlscoincidentwithslowerre-epithelialization,butneutrophil levelsarenotdifferentbetweenmorphs. L-plastinwasusedasapan-leukocyticmarkertoquantifytotalleukocytelevelsfollowinginjury.A) Totalleukocytescountedpermm 2 inthewoundbedat1,3,7and14dpi(n=4foreachmorph).L-plastinpositivecellsarered(yellowarrows),nuclei arestainedblue(Hoescht)andgreenfluorescencewasusedtoaccountforautofluorescencingerythrocytesthatwereexcludedasleukocytes.An influxofleukocyteswasobserved24hrsdpi,withhighernumberspresentinterrestrialaxolotls.LeukocytelevelsdroppedinmetamorphsatD7and convergedwithpaedomorphlevels.LevelsforpaedomorphswerenotsignificantlychangedafterD1.B)Allneutrophilspresentinthewoundbed (yellowarrows)werecountedon5 m msectionsabovethemuscleusingmyloperoxidase(n=8foreachmorph;seeFigureS6forpositivecontrol staininginliver).Neutrophillevelsweregenerallylowandwerenotsignificantlydifferentbetweenpaedomorphsandmetamorphs,althoughthey diddropsignificantlyatD7.C)ModifiedWright-Giemsastainusedtoindentifyindividualleukocytesincirculatingaxolotlblood.Sudanblackwas usedtostainneutrophils.Yellowarrowsindicatethespecificleukocytetype. doi:10.1371/journal.pone.0032875.g007 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org9April2012|Volume7|Issue4|e32875

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Figure8.Contractionduringscar-freehealinginaxolotlsissimilartotight-skinnedmammals. A)Percentwoundclosurein paedomorphsandmetamorphsover21days(whencontractioniscomplete).Metamorphwoundsexpandedby10%followingwoundingand woundscontractedataboutthesamerateinbothmorphs.B)Paedomorphwoundscontracted 27%morethanmetamorphwoundswith contractionaccountingfor37.9%ofwoundclosureinmetamorphs.C)Alpha-smoothmuscleactin(alpha-SMA)localizationinunwoundedskinand duringwoundrepairin Musmusculus andaxolotls( A.mexicanum )toidentifymyofibroblasts.Alpha-SMAlocalizedtobloodvesselsandafewcellsin mouseskinandaroundglandsandthestratumcompactuminaxolotlskin.Tendayspostinjury(dpi),whencontractionratesarehighestinmouse wounds,alpha-SMApositivecellsaredetectedathighlevelsatthewoundmargins(whitedottedcircle).Incontrast,wedetectedveryfewalpha-SMA positivecellstendaysafterwoundinginaxolotltissue.Wedid,however,detectalpha-SMAintheregeneratingbasementmembrane(yellowarrows). Twenty-onedpiwedetectedahighnumberofmyofibroblastsinmousetissue(redarrows).WedetectedafewmyofibroblastsinaxolotltissueatD21 (redarrows)neartheunderlyingmuscle. doi:10.1371/journal.pone.0032875.g008 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org10April2012|Volume7|Issue4|e32875

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appearedpositiveforalpha-SMA(Figure8C).Weobserved numerousalpha-SMApositivecellsatD21inmousegranulation tissuecomparedwithrelativelyfewinaxolotlprovisionalmatrix. Takentogether,thesedatashowthatwoundcontractionin axolotlsmimicwoundcontractioninhumanskin,althoughthere arerelativelyfewmyofibroblastspresentduringaxolotlskin regeneration. DermalregenerationisCharacterizedbyHighLevelsof Tenascin-C Theprocessofdermalrepairinmammals(whichultimately resultsinascar)proceedsviaproductionofafibrinclot,(referred toastheprovisionalextracellularmatrix),degradationand replacementofprovisionalmatrixbygranulationtissue,and remodelingofgranulationtissueintoafibroticscar[1].Following re-epithelializationinpaedomorphs(D1)andmetamorphs(D3), thewoundbedwasrichinbloodcellsandplasmabutnoscab formed(Figure2).Provisionalmatrixinmammalsisrichin fibronectinandthrombospondin[1]andalongwithplasmawe detectedlowlevelsoffibronectinatthewoundmarginsand beneaththewoundepidermisatD7inbothmorphs(Figure9A). Incontrast,wewereunabletodetectappreciablecollagen depositionatD7usinghistochemicaltechniques(Figure1and Figure3).Duringmammalianwoundhealing,depositionof granulationtissuematrixproceedsinthestereotypicalsequenceof fibronectin,collagentypeIIIandcollagentypeI[42].AtD14 fibronectinwaspresentinlowconcentrationsingranulationtissue inbothmorphs(Figure9A).Usingpicrosiriusredtodetectboth collagentypeIIIandcollagentypeI,wepredominantlyobserved collagentypeIIIbeneaththeepidermisatD14(Figure10).As collagensynthesiscontinuedcollagentypeIIIwasreplacedwith collagentypeIandasnewdermisbecameprogressivelyacellular, thedensityanddiameterofcollagentypeIfibersincreased (Figure10).Fibronectinremaineddetectableingranulationtissue (D21),albeitatverylowlevels(Figure9A). Whilehighlevelsoffibronectinandcollagenarepresentin granulationtissueandalsofibroticscartissue,theappearanceof tenascin-C(TN-C)duringmammalianwoundhealingisnormally restrictedtothewoundmarginsandthoughttostimulatecell migration.Concomitantwiththeseobservationswefoundintense TN-Cproteinlocalizationatthewoundmarginsandbeneaththe epidermisatD7(Figure9B).However,asdermalregeneration proceeded,TN-Cpersistedinhighamountsthroughoutthe woundbedandwithinregeneratingmuscle(Figure9B).This associationwasparticularlystrikingintheunderlyingmuscle whereTN-Cformedasharpboundarybetweenregeneratingand undamagedmuscle(Figure9B).TN-Clevelsremainedhighuntil thedermishadregenerated(Figure9B).Takentogetherthese findingssuggestthatcollagensynthesisduringaxolotlscar-free healingproceedssimilarlytothatwhichisobservedduring mammalianwoundrepairandlowlevelsoffibronectinand persistenthighlevelsofTN-Ccharacterizetheanti-scarring matrix. Discussion Althoughmanysalamandersarecapableofcompleteappendageregeneration(e.g.limb,tail),arecurringquestioniswhether woundsmadeonthebodyhealwithascarorinstead,healscarfree[32].Inthisstudywedemonstratecompleteandperfect regenerationofadultaxolotlskinfollowingfullthicknessexcisional (FTE)woundingtothedorsalflank.Additionally,wetestedthe hypothesisthatlossoflarvalskincharactersinmetamorphic axolotlsresultsinscarringfollowingFTEwounding.Instead,we findthatmetamorphicaxolotlsarecapableofscar-freehealing.To ourknowledge,thisisthefirstdemonstrationofperfectscar-free healingofnon-limbFTEwoundsinanaquaticorterrestrialadult vertebrate.Comparingskinregenerationinpaedomorphic (aquatic)andmetamorphic(terrestrial)axolotls,metamorphic axolotlsexhibitedslowerre-epithelialization,increasednumbersof leukocytesduringtheearlyinflammatoryresponse,increased depositionofextracellularmatrix(ECM)andanalmostdoubling inthetimerequiredforcompleteskinregeneration.Comparedto mammalianwoundrepair,terrestrialaxolotlsexhibitedareduced hemostaticresponse,lowerneutrophilslevels,similardurationof inflammation,fastertimetocompletere-epithelialization,adelay innewtransitionalmatrixproductionanddifferencesinthe relativecompositionofthenewECM(Figure11).Thesedata suggestthatfurtherexplorationofFTEwoundinginaxolotlsisan excellentmodeltoinvestigatethecellularandmolecular regulationofscar-freehealing. Hemostasis Woundhealinginmammalsproceedsalongastereotypical timelineconsistingofrepairprocessesthatoverlapintimeand spaceandtheseprocesseshavegenerallybeenincludedwithin threegeneralphases;inflammation,newtissueformationand tissueremodeling[1,6,43].Woundhealingbeginsimmediately afterinjurythroughtheextravasationofbloodproductsand activationofthecoagulationcascade.Inmammals,subsequent hemostasisisachievedthroughformationofathickclotformed fromplateletsandplasmaderivedfibrin.Thisclotactsasa temporaryplugandasareservoirofgrowthfactors,whilethe fibrinmatrixactsasascaffoldforinfiltratinginflammatorycells.In aquaticaxolotls,thehemostaticresponseappearedlimitedwitha thinlayerofcoagulatedplasmaatthewoundsitethatwasmostly devoidofcells.Followingre-epithelialization,theneoepidermis wasincloseproximitytotheunderlyingmusclewithlittleevidence ofapersistentfibrinclotpriortonewECMproduction.The presenceofaclotinterrestrialaxolotlswasmorepronounced,but wasstillgreatlyreducedcomparedtomammals.Erythrocytesand plasmaformedthemajorityofmaterialabovethefragmenting muscleanditwasuncleartowhatextentfibrincontributedtothe looselyaggregatedclotmatrix.Followingre-epithelialization,some bloodplasma,erythrocytesandleukocyteswereobservedabove andwithinthefragmentingmusclefibers,buttheextentofaclot remainedminimalandtheepidermisremainedincloseproximity totheunderlyingmuscle.Takentogether,thesefindingsshowthat axolotlsrestorehemostasisquicklyandwithoutformationofan extensivefibrinclot. Duringmammalianwoundhealing,plateletsdegranulate, initiatethecoagulationcascadeandprovideawealthofgrowth factorsandchemokines(e.g.PDGF,TGF§,VEGF,EGF,and IGF)thatactivatemesenchymalcellsandattractinflammatory cellstothewoundsite[7,44].Althoughsomeevidencesuggests thatplateletsarenotrequiredduringwoundhealinginmammals [45],allofthesemoleculeshavebeenshowntoplayaroleduring woundrepair[46].Thedegranulatingactionofamphibian platelets(thrombocytes)duringinjuryispoorlyunderstood,as arethegrowthfactorsproducedduringhemostasis.Futurestudies investigatingtherelativecomplementofchemoattractantsand mitogenspresentintheclotmatrixduringscar-freehealingin axolotlswillshedlightonhowthismolecularcocktailmightdirect theearlyeventsofwoundhealingtowardsregenerationinlieuof scarring. Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org11April2012|Volume7|Issue4|e32875

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Inflammation Includinghemostasis,thesequentialinfluxandactionof neutrophils,macrophagesandlymphocytesconstitutestheinflammatoryphaseofwoundrepair.Inmammalianwounds,neutrophilsarethepredominantcelltypepresentimmediatelyafter injuryandarenecessarytodestroybacteriaandcombatinfection. Therecruitmentofneutrophilsoccurspassively(throughplasma aggregation)andviachemotaxisinresponsetotheactivationof complement,degranulatingplatelets,andfrombacterialdegradation.Neutrophilsinturnhavethecapacitytoattractadditional inflammatorycellsandamplifytheinflammatoryresponse. Monocytesarriveatthewoundsite23daysafterinjuryand transformintomacrophages.Macrophagesservetoremove neutrophilsandcellulardebris,whichhelpstoresolveinflammaFigure9.RegenerativeECMinaxolotlwoundsischaracterizedbyhighlevelsoftenascin-C. A-B)Fibronectin(FN)andtenascin-C(TN-C) levelsweredetectedduringscar-freehealinginpaedomorphsandmetamorphsusinganantibodytoaxolotlfibronectinandapolyclonalantibodyto chicktenascin-C.WedetectedlowlevelsofFNinthebasementmembraneatD7,andatthewoundmarginsinbothmorphs.FNwaspresentduring ECMdepositionatD14inthecenterofthewoundbed,butinrelativelysmallamounts.ByD21littleFNpersistedintheregeneratingdermis.B)TN-C wasdetectedatthewoundmargins,inthebasementmembraneandsurroundingsomecellsatD7.Fourteendayspostinjurywedetectedhigh levelsofTN-Cthroughoutthewoundbedandinregeneratingmuscle.Asharpboundaryformedbetweenintactmuscleandregeneratingmuscle. ThesehighlevelsofTN-Cpersistedduringdermisregeneration.Greenfluorescencewasusedtodetectautofluorescingerythrocytes.Epidermis(E) dermis(D),muscle(M),woundmargin(WM). doi:10.1371/journal.pone.0032875.g009 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org12April2012|Volume7|Issue4|e32875

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tion,andalsoarethoughttoassistlaterduringnewtissue formationtoregulatethebalancebetweenfibrosisandscarring [36].FollowingFTEwoundinginaquaticandterrestrialaxolotls wedetectedneutrophils24hrsafterinjury.Neutrophillevels, however,werereducedcomparedtoequalsizeFTEwoundsin mammalssuggestingareductionthroughpassiveaggregationor chemotaxis[47].Comparedtomammals,whichmaintain 60% oftheircirculatingleukocytesasneutrophils,wefoundaxolotls maintainapproximately 21%oftheircirculatingleukocytesas neutrophilsandthissuggeststhatlowneutrophilnumbersatthe woundsiteresultpartlyfromlownumbersincirculation.In supportofthisfinding,arecentstudyinlarvalaxolotlsfoundvery fewneutrophilsinpartialthicknesstailwounds(whichincurvery littlebleeding),whilestabwoundsmadeintothetailmuscle,ledto greaternumbersatthewoundsite[26].Neutrophildepletion studiesinmammalshaveshownthataslongasconditionsremain sterile,neutrophilsarenotrequiredduringwoundhealing,and theirlossmayactuallyincreasetherateofre-epithelialization [48,49].Ourfindingsshowthattheirpresence,albeitinlow numbers,iscompatiblewithscar-freehealingandiscoincident withafasterrateofre-epithelializationcomparedtomammalian wounds. Althoughwedidnotexamineadditionalindividualleukocytic lineages,comparingL-plastinpositivecells(apan-leukocytic marker)inaquaticandterrestrialaxolotls,wefoundhighertotal leukocytenumberspersistinginterrestrialanimalswhilethe woundbedremainedexposed.Followingre-epithelialization,total leukocytenumbersconvergedforbothmorphssuggestingthatthe earlyinfluxofinflammatorycellsmaynotsignificantlyinfluence thesubsequentarrivalofinflammatorycells.Together,ourfinding thattotalleukocytelevelsremainhighatleast14dayspostinjury Figure10.CollagentypeIIIpredominatesearlyduringnewtissueformationandisslowlyreplacedbycollagentypeIduringscarfreehealing. PicrosiriusredstainingwasusedtodetectcollagentypeIIIandcollagentypeIduringscar-freehealinginthewoundbed.Using polarizedlighttodetectbifringence,collagentypeIII(greenfibers)wasdepositedfirstduringnewECMdepositioninbothmorphs.Asthedermis regenerated,collagentypeIIIwasslowlyreplacedbycollagentypeI(redfibers)inbothmorphs. doi:10.1371/journal.pone.0032875.g010 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org13April2012|Volume7|Issue4|e32875

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supportstheroleofanactiveinflammatoryphaseduringscar-free healinginaxolotls. Thenotionthatreducedinflammationpromotesregeneration inlieuofscarringhasbeenpopular,butevidencefrommammals andamphibiansremainsinconclusive[50,51].Aspectsoflimb regenerationinadultanurans,whichlosetheabilityforlimb regenerationfollowingmetamorphosis,canbestimulatedthrough tissueaggravation[52]andchemicalirritation[53],suggesting thatprolongingtissueinflammationcanprimeadormant regenerativeresponse.Ontheotherhand,scar-freehealingin fetalmammalsiscorrelatedwithreducedinflammationpriorto theirdevelopmentofamatureimmunesystem(comparedto scarringinadults)[10,54].Datafromadultmammalssuggests platelets[45],neutrophils[48]andmacrophages[55]are dispensableforwoundrepair,buttheirremovaldoesnotleadto scar-freehealingsuggestingthatreducinginflammationalonewill notinducearegenerativeresponse.Theexistingdatafromchronic andhypertrophicwoundssupportthehypothesisthattoomuch inflammationpromotesexcessivefibrosis,andinthecontextofour results,itislikelythatinflammationmustnotexceedathreshold levelforscar-freehealingtooccur[7,56].Arecentstudydesigned toincreaseinflammationinaxolotlsbyinjectingbleomycin followingpartialthicknesstailwounds,foundevidenceof increasedfibrosisfollowingthirtydaysofadministeringthedrug duringthehealingprocess[26].Whileitistemptingtospeculate thatthisexposuremightleadtoscarring,giventhecontinuous exposuretobleomycinpriortotissueharvestitisdifficultto interprettheirresultsastheyrelatetoheightenedinflammation earlyduringthehealingprocess.FutureexperimentsoverstimulatinginflammationinourterrestrialFTEmodelofscarfreehealingthroughchemicalandmolecularmethodsduringthe naturallyoccurringwindowofinflammationwillallowusto Figure11.Summaryofwoundhealingprocessescomparingaxolotlsandmammals. Thex-axisrepresentstimeandthey-axisrepresents percentmaximalresponseforeachprocess.Informationformammalshasbeenapproximatedfromtheliteratureandfromourownexperiments with4mmFTEwoundsinmice.Colorsrepresentindividualprocessesoverlappingacrossthethreephasesofwoundhealing;inflammation,new tissueformationandtissueremodeling.Comparingpaedomorphicandmetamorphicaxolotls,metamorphsexhibitedanincreasedhemostatic response,slowerre-epithelialization,increasedearlyinflammatoryresponse,increasedandprolongeddepositionofextracellularmatrix(ECM )andan almostdoublinginthetimerequiredforcompleteskinregeneration.Comparingscar-freehealinginterrestrialaxolotlstoscarformationinmamma ls, terrestrialaxolotlsexhibitedareducedhemostaticresponse,lowerneutrophillevels,fasterre-epithelializationrate,delayinECMproductio n, differencesintherelativecompositionofthenewECM,regenerationofglandsanddermisregenerationinsteadofscarring.Schematicformammals isadaptedfromMikaelHa ggstro m. doi:10.1371/journal.pone.0032875.g011 Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org14April2012|Volume7|Issue4|e32875

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examinethecellularandmolecularmechanismsthatconnect inflammationandfibrosis.Ourfindingsreinforcetheideathat inflammationisnotaltogetheranti-regenerativeandsupportarole foranactiveinflammatoryresponseduringscar-freehealing. Re-epithelialization Astheinflammatoryphasebeginstowinddown,keratinocytes atthewound-edgemigratetore-epithelializethewoundbedand restorethephysicalbarrierseparatingtheexternalenvironment. Concomitantly,granulationtissue(transitionalmatrix)isproduced toreplacethefibrinclotandtogethertheseprocesseswill contributetonewtissueformationatthewoundsite.Wefound thatadultpaedomorphicaxolotlscompletelyre-epithelialized 4mmwounds1824hrspostinjury,whileterrestrialaxolotls exhibitedslowerre-epithelialization.Interrestrialaxolotlskin, woundedgekeratinocytesmigratedafteradelayperiodof 24hrsandcompletere-epithelializationoccurredupto3days afterinjury.Thisfindingsuggeststhatthetransitionfroma pseudo-stratifiedtostratifiedepidermisfollowingmetamorphosis preventsrapidre-epithelializationinresponsetoinjury.Previous researchinnewtlimbssuggestedthatepidermalkeratinocytesare constitutivelyactiveandthusareprimedtomigratefollowing injury[24].Ourfindinginpaedomorphssupportsthisconclusion, whiletheobserveddelayinterrestrialaxolotlsdemonstratesthe needforkeratinocyteactivationasoccursinmammalianwound healing[57].Interestingly,theonsetofkeratinocytemigrationin mammalsoccursafteradelayperiodof1824hrs[57,58],which isidenticaltowhatweobserveinterrestrialaxolotls.FTEwounds madeinfetalratsthathealscar-free(E16),orresultinscarring (E19),bothre-epithelializein72hrsandtogetherwithourdatain terrestrialaxolotls,suggestthatthespeedofre-epithelialization cannotaloneexplaintheabilitytoperfectlyregenerateskin[13]. Ourresultssuggestthatrapidre-epithelializationreducesthetime requiredtoregenerateskin,andmaycontributetoreduced inflammation.Onceactivated,terrestrialaxolotlkeratinocytes appearedtomigraterapidlyacrossthewoundbedanditwouldbe interestingtocomparetherateofre-epithelialmigrationtosimilar sizedmammalianwoundswheremoisturepreventsscabformation.Comparingepidermalcellsbetweenthesetwomorphsduring woundhealingwillprovideapowerfulmodeltoidentifythe molecularcontrolofkeratinocyteactivationandwillbeusefulin identifyingnewmoleculesthatmightstimulatekeratinocyte migrationinnon-healingchronicwounds. ECMProductionandNewTissueFormation Whileadampenedhemostaticresponse,reducedinflammation andquickerrateofre-epithelializationlikelycontributestoscarfreehealinginaxolotls(comparedtoscarringinadultmammals), theslowdepositionofnewextracellularmatrix(ECM)andits uniquemolecularcompositionsuggestsnewlysynthesizedaxolotl ECMmayantagonizefibrosisandpromoteregeneration (Figure11).Shortlyafterre-epithelializationbeginsduring mammalianwoundrepair,theprovisionalmatrix,whichisrich incross-linkedplasmafibronectin,isreplacedbygranulationtissue richinmacrophages,fibroblasts,newlysynthesizedECM moleculesandneovasculature[59].Withinthisgranulationtissue, fibroblastssecretefibronectin,collagentypeIIIandcollagentype I,andthismatrixwillformthetransitionalECMthatwill ultimatelyberemodeledintodermalscartissue[1,60].Although fibronectinandcollagenarethemostabundantECMmoleculesin mammaliangranulationtissue,tenascin-Cisalsodeposited,firstat thewoundmargins,thenattheepidermal-dermaljunctionof migratingkeratinocytes,andfinally,withinthegranulationtissue itself[61].Duringscar-freehealinginterrestrialaxolotlsthe woundbedattheepidermal-dermaljunctioncontainedlittlenew ECMfor10daysafterre-epithelialization.Thisdelayperiodwas strikinginitsdurationandourfindingthattheneoepidermiswasa potentsourceofseveralmatrixmetalloproteinases(MMPs) suggestsanactiveinhibitionofnewECMproduction.Whilewe foundstrongandrapidupregulationofseveral MMPs ( MMP1, axCol,MMP3/10a,b,MMP19,MMP28,MMP9 )priorto,and duringre-epithelialization,mostofthesereturnednearbaseline levelsfollowingre-epithelializationsupportingtheirroleduring keratinocytemigration[25].However, MMP3/10b and MMP9 persistedatrelativelyhighlevelsintheneoepidermis,and MMP2 levelsbeganincreasingafterre-epithelialization.Allthreeofthe homologousmammalianproteinasescandegradefibronectin,and MMP3andMMP2arecapableofdegradingcollagentypeIIIand typeI[62,63].Togetherthesedatasuggestthatduringscar-free healinginaxolotls,persistentlyhighMMPlevelsmayactafterreepithelializationtopreventnewECMproductionasresidenttissue undergoeshistolysisandthewoundbedispreparedfordeposition oftheregenerativematrix.Supportingthisnotion,broad inhibitionofMMPsduringnewtlimbregenerationcanleadto acellularscartissueformationandstudiesduringscar-freehealing infetalwoundshaveobservedhigherMMPlevelscomparedto adultwounds[13,34].Ongoingstudiesinourlaboratoryare targetingtheseindividualMMPsandtheirspecificactivities towardsunderstandinghowtheyregulatebothre-epithelialization andthefibroticresponseduringscar-freehealing. TheroleofotherproteinasessuchastheADAMsand ADAMTSsduringmammalianwoundhealingispoorlyunderstood,whiletheirroleduringfetalwoundhealingiscompletely unknown.Theirabilitytocleavemembrane-bounddomainsofa hostofdifferentproteins(e.g.cytokines,growthfactors,cytokine andgrowthfactorreceptors)thusreleasingthemintothe extracellularspace,hasdrivenspeculationthatthesefactorsmay playanimportantroleduringwoundhealing,althoughdirect studiesarelacking[64].OnlyADAM9andADAMTS1havebeen foundtohaveadirectroleduringwoundhealing,withfasterreepithelializationoccurringinmouseknockoutstudiesofboth individualgenes[65,66].Duringscar-freehealinginaxolotlswe foundasmallbutsignificantincreasein ADAM9 followinginjury butdidnotdetectchangesabovebaselineinanyoftheother ADAM or ADAMTS genes.Together,ourfindingssuggestthat ADAMsmaynotplayavitalroleduringscar-freehealing, althoughtheyarelikelytoworksynergisticallyduringkeratinocyte migration. ComparingourECMdatawiththatacquiredfromstudieson scar-freehealinginfetalmammals,bothsimilaritiesand differencescanbenoted.Althoughtherehasbeenconsiderable controversysurroundingtheproductionofcollagensinfetal wounds,owingtothevarietyofspecies,woundmodelsandfetal age,itisgenerallyacceptedthatcollagensareproducedduring fetalwoundhealingandareproducedmorerapidlycomparedto adultwounds[67,68].Whilecollagenfibrilsaredepositedindense bundlesparalleltothewoundbed,fetalcollagenfibrilsappearto bedepositedinareticularfashion,almostidenticaltothe surroundingdermis[68].Thiscontrastswiththesituationduring scar-freehealinginaxolotlswherecollagendepositionwasdelayed (asinadultmammals)andalsooccurredindensebundles throughoutthewoundbed.Thusourdatasuggeststhatthe patternofcollagendepositionobservedduringadultscarringisnot technicallyincongruentwithregenerationandmaysuggestthe lackoflaterremodelingeventsordifferencesinthestructural compositionofthecollagendeposited. TheappearanceofnewECMduringaxolotlwoundhealingwas coincidentwithfibroblastinfiltrationbeginningatD14,andsmall Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org15April2012|Volume7|Issue4|e32875

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amountsoffibronectinproduction.Similarly,fibronectinis recognizedasaprominentcomponentoftheprovisionalmatrix duringadultandfetalwoundhealing,appearingearlyassociated withthefibrinclotandthenslowlydisappearingconcomitantwith thedepositionofcollagen[60,68].Itisexpressedearlierinfetal woundscomparedtoadultwoundsanditsdisappearanceis correlatedwiththeonsetofcollagendeposition.Itisalsopresentat highlevelsinunwoundedfetalskincomparedtoadults[69].In contrasttomammalianwounds,axolotltransitionalmatrixwas highlyenrichedwithtenascin-C.Fibronectinappearedtransiently withcollagentypeIIIinthewoundbedwhiletenascin-Cpersisted throughD42ascollagentypeIwasproducedandthedermis regenerated.Theroleoftenascin-Cduringfetalwoundhealing hasbeenpoorlystudied,althoughinthesamestudythatexamined fibronectindistributionduringincisionalhealinginthelip[68] tenascin-Clevelswerefoundtobeslightlyhigherinthefetalversus theadult.Somedatasuggeststhattenascin-Ccanantagonize certainfunctionsoffibronectinincludingT-cellactivation[70] andinthecontextoffetalscar-freehealingandaxolotlscar-free healingsuggeststhattherelativeconcentrationofeachinthe extracellularmatrixmightacttoregulateanadaptiveimmune response.Tenascin-Cisthoughttobehaveasananti-adhesive moleculepromotingproliferationandmigrationoverdifferentiationandassuch,isatransientmoleculeduringmammalian woundhealing[1].Tenascin-Ccandisruptcell-matrixinteractionsandhasbeenspeculatedtomaintainmonocytesand blastemalcellsinade-differentiatedstateduringsalamanderlimb regeneration[23,71,72].Thesedatasuggestthatthecomposition ofECMproducedduringaregenerativeresponsemayhavean anti-fibroticeffectthathelpstorecapitulateembryonicdevelopmentandpromotearegenerativeresponse. RemodelingandRegeneration Theevolutionaryprocessthathasledtowoundrepairwhile efficient,isnotfunctionallyperfect.Epidermalappendagesdonot regenerateandtheuninjureddermalarchitectureisreplacedby denseparallelbundlesofcollagenthatreducethemechanical propertiesofnormalskin.WhilethewoundbedECMduring mammalianwoundrepairwilleventuallyresultinafibroticscar, weobservedcompleteregenerationofnotonlydermallayersbut ofepithelialderivedglands.Theregenerationofhairfollicleshas beenobservedinrabbitwoundsandearpunchesandinverylarge woundsmadeonyoungmice[73,74,75].Otherthanthesereports, theregenerationofepithelial-derivedstructuresinadultvertebrateshasnotbeenobserved.Althoughhairfollicleandgland developmentiswellunderstoodinmammals,itispoorly understoodatthemolecularlevelinamphibians.Ongoing experimentsinourlaboratoryexaminingthemolecularcontrol ofglanddevelopmentareunderwayanditwillbeinterestingto determinehowregenerationofthesestructuresiscontrolled followingscar-freehealing[76].Specifically,itwillbeofinterestto determinethelocationofinductivesignalsthatleadtothe specificationofnewglandsandwhetherthesesignalsarelocalized orubiquitousduringtheregenerativeprocess. AxolotlModelofScar-freeHealing Thedatainthispaperestablishesanovel,adultmodelofperfect skinregenerationinwhichtotesthypothesesaboutthemolecular regulationoffibrosisandmoregenerally,theunderlyinggenetic controlofscar-freehealing.Assuch,wehavedevelopedanFTE woundmodelinbothaquaticandterrestrialaxolotlsand characterizedtheirabilitytoperfectlyregeneratealldamaged tissues.Todateno adult animalmodelhasbeendescribedthatis capableofperfectlyhealingFTEwoundsandunderlyingmuscle (outsideofthelimbandtail).Whilenumerousstudieshave describedhealingwithoutscarringinfetalmammalsand marsupials[10],thefetalmodelprovidesanumberofdrawbacks thattheaxolotlmodelovercomes.First,thefetusisstilldeveloping inthewombandthiscreatesbothbiologicalandpractical complications.Thedevelopingfetus,atthetimewhenitheals scar-free,hasanimmatureendocrinesystem,isimmunoincompetent,iscontainedinamoiststerileenvironment,andits cellsareinastateofchronichypoxia[16].Incontrasttofetal models,terrestrialaxolotlsarefullydeveloped,sexuallymature, haveafullydevelopedimmunesystem(albeitnotassophisticated asadultmammals),andtheirFTEwoundsaredesiccatingand opentoinfection.Fromapracticalstandpoint,inutero''surgery isnotrequired,uptosix4mmFTEwoundscanbemadeonthe dorsalskinandpharmacologicdrugsandchemicalscanbeeasily deliveredtoantagonizeoragonizesignalingpathways.In combinationwiththeirabilitytoperfectlyregeneratealltissues damagedinthesewounds(e.g.epidermis,dermis,glands,nerves, muscle)thismodelshiftsexperimentationintoasystemwherethe ultimateclinicaloutcomeactuallyoccurs. Thisnewanimalmodelwillprovideinvestigatorsanew paradigminwhichtoaddressthemultipleprocessesofwound repair(e.g.hemostasis,inflammation,keratinocyteactivationand migration,ECMformation,tissueremodeling)inatrulyscar-free healingadult,whilealsoprovidingatractablegeneticsystemto investigatethecellularandmolecularmechanismsthatregulate theseprocesses.Giventheconsiderablebodyofliteratureonthe molecularpathwaysandcellularprocessesinvolvedduringnormal woundrepair,understandinghowtheseconservedmolecular playersoperateinapro-regenerativeenvironmentwillleadto developmentofnewtherapiesthattargetdownstreameffectorsfor testinginestablishedmammalianmodels. MaterialsandMethods AnimalsandWoundModel EthicsStatement. Allprocedureswereconductedin accordancewith,andapprovedby,theUniversityofFlorida InstitutionalAnimalCareandUseCommittee(IACUCStudy # 201101534and # 200903505). Animals. Ambystomamexicanum (Mexicanaxolotl)were acquiredfromtheAmbystomaGeneticStockCenter(AGSC, LexingtonKY)andbredincaptivity.Animalswerehoused individuallyinAquaticHabitats H (AquaticHabitatsInc.,Apopka, FL)flow-throughsystemsat2123 u CinHoltfreter'sSolutionand maintainedonCaliforniablackworms( Lumbriculussp .,J.F. Enterprises,Oakdale,CA).Animalsweredeemedadultsafter reachingsexualmaturity( 9months)andmeasuredbetween12 15cmtotallength.Metamorphic(terrestrial)axolotlswere acquiredeitherdirectlyfromAGSCortransformedatthe UniversityofFloridathroughtreatmentwiththyroxine[77].In bothcasesmetamorphosisoccurredafteranimalshadreached sexualmaturity.Terrestrialaxolotlswerehousedindividuallyin 10-litercontainerscontainingmoistpapertowelsandfed nightcrawlers.MiceusedinthisstudywereSwissWebster (CharlesRiver)approximately6-monthsold. Fullthicknessexcisional(FTE)wounding. Adultaxolotls wereanesthetizedbyfullsubmersioninBenzocaine(Sigma)0.01% (aquatic)or0.02%(terrestrial).Sterile4mmbiopsypuncheswere usedtocreateFTEwoundsthroughtheskinintothedorsalback muscle.Inthisway,6woundswerecreatedonthedorsalsurface posteriortotheforelimbsandanteriortothehindlimbs.Inorder toharvestwoundtissueatspecifictime-points,animalswere anesthetizedasaboveandtheentirewoundwasharvestedusinga Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org16April2012|Volume7|Issue4|e32875

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6mmbiopsypunchandiridectomyscissors.FTEwoundsinmice (throughthepanniculuscarnosus)weremadewith4mmbiopsy punchesthroughthedorsalskinposteriortotheforelimbsand anteriortothehindlimbs. HistologyandImmunohistochemistry Forhistologicalandimmunohistochemicalanalysis,harvested tissueswerefixedin10%NBFat4 u Cfor1624hr,washedinPBS anddehydratedto70%EtOH.Sampleswerestoredat4 u Cand processedforparaffinembedding.Sampleswerecutat5 m m.For frozensections,sampleswerefixedfor1hrin10%NBFat4 u C, washedinPBS,equilibratedinsucroseandOCTfreezingmedium andfrozeninOCT.Frozensectionswherecutat12 m m.For histologicalanalysis,paraffinsectionswerestainedwithoneofthe following:Masson'sTrichrome(RichardAllenScientific)or PicrosiriusRed(AmericanMastertech).Forbloodsmears,blood wascollectedfromlimbamputationsandair-driedonslides.Slides werestainedwithSudanBlack[78]todetectneutrophilsand counterstainedwithamodifiedWright-GiemsaStain(Kodak). Immunohistochemistry. Slideswererehydrated,antigen retrievalperformedifnecessary(seebelowforeachantibody), washedinTBS,blockedforstreptavidinandbiotin(VectorLabs), andincubatedwith1 u antibody(seebelow)overnightat4 u C.The followingday,slideswerewashedinTBS,incubatedwith biotinylated2 u antibody(VectorLabs)andsubsequently incubatedwithstreptavidinconjugatedhorseradishperoxidase antibody(VectorLabs)andvisualizedusingDAB(3,3 diaminobenzidine)(VectorLabs)orincubatedwitha streptavidinconjugatedAlexa-Flour594(Invitrogen).Images werecapturedonaNikonEclipse6600uprightcompound microscopeusingaCool-SnapProtruecolorcamera(light microscopy)oranOlympusinvertedmicroscope(IX81)with fluorescenceusingaLeicaDFC310FX(fluorescencemicroscopy). Antibodies. Forparaffinsections;WidespectrumCytokeratin (DAKO;Z0622)1:500, Ambystoma Fibronectin(giftofThierry Darribere)1:1000,Tenascin-C(Millipore,AB19013)1:500, CollagentypeIV(Rockland,600-401-106-0.1)1:450,alphasmoothmuscleactin(Abcam,ab5694)1:100,Laminin(DAKO, Z0097)1:500.Forfrozensections;Myloperoxidase(Thermo,RB373-A)1:100,L-plastin(giftofPaulMartin)1:2000.Antigen retrievalsusedwere;microwaveandCitrateBuffer(pH.6.0) (Cytokeratin,Myloperoxidase,L-plastin,Tenascin-C,alphasmoothmuscleactin)orProteinase-Ktreatment(DAKO)for 2mins(CollagentypeIV,Fibronectin,Laminin).Negative controlsusedisotypedIgGatthesameconcentrationasthe1 u antibody.AGFPfilterwasusedtodetectautofluorescenceof erythrocytes. MicroarrayAnalysis Epidermaltissuewasharvestedusinga4mmbiopsypunch. Twowoundsweremadealongtheflankandposteriortothe forelimbs.Harvestedepidermiswaspooledforeachanimal.Four biologicalreplicateswerecollectedfromuninjuredepidermis(D0) andat1,3,and7dayspostinjury.RNAwasisolatedusingTrizol Reagent(Invitrogen)followedbyQiagenRNeasyClean-up columns(Qiagen).RNAqualitywasassessedonanAgilent2100 Bioanalyzer(Agilent).RNAprocessingforAffymetrixmicroarray analysiswasperformedattheUniversityofKentuckyMicroarray CoreFacilityandhybridizedto32,AMBY_002a520748F2 nd GenerationAxolotlAffymetrixmicroarrays(Affymetrix)[79]. Microarraydatawastestedforqualitycontrolandanalyzedin thesoftwaresystemRusingtheBioconductorpackageand oneChannelGUI[80,81,82].Microarraydatawaspreprocessed, andnormalizedusingRMAandalinearmodeltestingtheeffectof timeforeachmorphtypewasfitforeachgeneacrossall32 GeneChipsusinglimma[83,84].Thus,twogenelistswere producedcontainingsignificantlychangedgenesovertimein paedomorphicormetamorphicanimals.Intheseanalyses, contrastsweremadebetweeneachtimepointandat-statistic wasproducedforeachcontrast.ThemoderatedF-statistic calculatedbytheeBayeslimmafunction[85]wasusedasan overalltestforsignificanceacrosstime,similartoANOVA.Agene wasconsideredstatisticallysignificantifithadanFDRadjustedpvalue 0.05andchangedatleast2foldbetweenanytwotime points.ThemicroarraydataisMIAMEcompliantandtheCEL filesareavailableonSal-Site(www.ambystoma.org).Inaddition, therawdataisavailablethroughthepublicNCBIGEOdatabase (accessionnumber:GSE35255). Statistics Forquantificationofinflammationweuseda2-wayANOVAto examinetheeffectsofmorph(metamorphversuspaedomorph) anddaypostinjuryontotalleukocytenumbers(withindividualas arandomeffecttoallowforrepeatedsampling).Forallother statisticalanalysesweusedaStudent'sT-test. SupportingInformation FigureS1 Axolotl(paedomorph)skinglands. A)Granular gland.B)Mucousgland. (TIF) FigureS2 Detailedaspectsofdermisandglandregeneration. A)Highmagnificationimageofaglandregenerating anddescendingfromtheepidermis44dpi.B)Detailofwound margin(WM)showingtheedgeoftheinjuredstratumcompactum whichisnormallydenselycompactedandtheloosecollagenfibers beginningtocoalesceinthewoundbed.C)Detailofwoundbed 180dpishowingmaturemucousglandandregeneratedstratum compactum.Hypodermis(H)anddermis(D).Scalebars= 100 m m. (TIF) FigureS3 Detailedaspectsofmetamorphicaxolotlskin regenerationover147days. A)Granularandmucousglands (yellowarrows)presentinthestratumspongiosum.Collagenfibers arepresentbetweentheglands.B)Somecellularaggregationsin theepidermisappeartobeearlystagesofregeneratingglands (yellowarrows).C)Regeneratedglandswithindenselycompacted collagenousECMbeneaththeepidermis.D)Stratumcompactum isbeginningtocoalesceastherestofthedermishasregenerated. Somefibrotictissueremainswithintheregeneratedmuscle.E) Completescar-freeskinregenerationatD147dpi.Alltissuelayers arepresent.Granularglandsremainimmaturecomparedto uninjuredskin.Scalebars=100 m m. (TIF) FigureS4 Visualizingtherateofre-epithelializationin paedomorphicaxolotlsusingGFPtransplantedskin. 1.5cm 6 1.5cmsquaresofdermisandepidermisfromubiquitouslyexpressingGFPaxolotlsweretransplantedtosamesize explantedareasonthetailofadultwhiteaxolotls.Aftertransplants hadhealed4mmFTEbiopsypunchesweremadethroughthe transplantedtissueandGFP-labeledepidermiswasobservedand photographedmigratingtocoverthewoundbedover18hrs. Migrationwasobservedbeginning3hrspostinjuryandwas completebetween18and24hrs. (TIF) Scar-FreeSkinRegenerationinAdultAxolotls PLoSONE|www.plosone.org17April2012|Volume7|Issue4|e32875

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FigureS5 MMP transcriptionandactivityarecorrelatedduringtailregenerationinpaedomorphs. A)Axolotl MMP9 expression(semi-quantitativePCR)fromtailtissue followingamputationandthroughD14post-amputation.Expressionisdownregulated7dayspostinjury.B)Gelatinzymography wasusedtoassessMMPactivityover21dayspostinjury.Red arrowpointstotentativelyassignedMMP9positionbasedonsize ( 85kDa)frompreviouslypublishedworkinnewtandaxolotl (Vinaraskyetal.2005,Santoshetal.2011).C)Gelatinaseactivity lagsbehindtranscriptionalupregulationandpeaksatD7.For quantificationofactivity,gelimageswereinverted,meanoptical densityofindividualblackbandswerecalculatedonanGelLogic gelimagingsystem(Kodak)andlightwasblockedtocalculatea baseline(black)referenceforeachmeasurement.Standarderrors arereported.Regeneratingtailtissueandtissue0.5mmrostralto theamputationplanewascollectedat0hrs,3hrs,6hrs,12hrs, 24hrs,7d,14d,and21dafterinjury(n=3pertimepoint),snap frozenondryice,andstoredat80 u C.Tissueswerehomogenized for10minutesonicein1:4(w:v)homogenizationsolution [50mol/LTris-Cl(pH7.6),150mol/LNaCl,1%(v/v)TritonX100,10mol/LEDTA,1mol/LPMSF],sonicated,setonicefor 10minutes,andspunat14000 6 g for10minutesat4 u C. Supernatantsweredecantedoff,quantifiedusingaBCAprotein assaykit(Pierce),andstoredat80 u C.25 m gtotalproteinwas dilutedinzymogramsamplebuffer(Bio-Rad)andelectrophoresed onReadyGelzymogrampolyacrylimidegelscontaininggelatin (Bio-Rad).MMPproteinswerere-naturedbywashinggelsin zymogramrenaturationbuffer(Bio-Rad)for30minutesand incubatedfor16hrsat37 u Cindevelopmentbuffer(Bio-Rad). Gelswerestainedwith0.5%(w/v)CoomassieBlueR-250(BioRad)for30minutesandde-stainedwithanaceticacid,methanol, anddH20solution(1:5:4)untilclearbandswerevisible. (TIF) FigureS6 Individualvariationinleukocytenumbers duringscar-freehealing. A)Totalleukocytenumbersbased onL-plastinstainingforpaedomorphsandmetamorphs(n=4for eachmorph).Becausemultiplewoundsweremadeonthesame animalstheinflammatoryresponsecouldbetrackedper individual.B)Onepaedomorphexhibitedanunusuallyhigh inflammatoryresponse(paed4)andweremoveditfromour analysis.C)Variationacrossmetamorphsrevealednooutliers.D) Controlstainingformyloperoxidaseinaxolotlliversection. ( ) TableS1 Axolotlgeneidentificationsbasedonfirst homologoushumangenehitinBLAST. Expressionvalues reflectabsoluteexpressionongenechip(maxexpressionvalue=30k;expressionvalues 100=noexpression).N=4animals pertimepoint.D0P=day0paedomorph,etc.D0M=day0 metamorph. (XLS) TableS2 Leukocyteprofilesfor Ambystomamexicanum (paedomorphandmetamorph). Numbersforeach particularleukocytetyperepresentedasapercentageoftotal leukocytes(thrombocyteswerenotcounted). (DOC) Acknowledgments WethankSriPuttaforhelpwithbioinformatics,MardaJorgensonfor histologicalassistanceandAshleyStuartforanimalcare. AuthorContributions Conceivedanddesignedtheexperiments:AWSMM.Performedthe experiments:AWSJRMMM.Analyzedthedata:AWSJRMSRVMM. Wrotethepaper:AWS.Commentedandeditedthemanuscript:JRM SRVMM. 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