CharacterizationofTGFβsignalingduringepimorphictissueregeneration:anexampleusingtheleopardgecko(Eublepharismacularius)tailregenerationmodel.

by

RichardW.D.Gilbert

AThesisPresentedto

TheUniversityofGuelph

InpartialfulfillmentofrequirementsforthedegreeofMastersofScience

inBiomedicalSciences

GuelphOntario,Canada

©RichardW.D.Gilbert,February2013

ABSTRACT

CharacterizationofTGFβsignalingduringepimorphictissueregeneration.An

exampleusingtheLeopardGecko(Eublepharismacularius)tailregenerationmodel.RichardWilliamDonaldGilbert Advisors:UniversityofGuelph,2013 Dr.A.M.Viloria‐Petit Dr.M.K.Vickaryous

Thetransforminggrowthfactorbeta(TGFβ)/activinsignalingpathwayhasa

numberofdocumentedrolesduringwoundhealingandisbecomingincreasingly

appreciatedasavitalcomponentofmulti‐tissueregeneration.Theleopardgecko

(Eublepharismacularius)isabletospontaneously,andrepeatedly,regenerateitstail

followingtailloss.Wethusexaminedtheexpressionandlocalizationofseveralkey

componentsoftheTGFβ/activinsignalingpathwayduringtailregenerationofthe

leopardgecko.Weobservedamarkedincreaseinphosphorylated‐Smad2

expressionamongregeneratingtissuescorrespondingtothelocationofthe

regenerateblastema.Interestingly,weobservethatduringearlyregenerationthere

appearstobeanabsenceofTGFβfamilymemberTGFβ1andinsteadastrong

upregulationofactivin‐βA.WealsoobservetheexpressionofEMTtranscription

factorsSnail1andSnail2inblastemaltissue.Theseobservationscombinedwith

otherdataprovidestrongsupportfortheimportanceofuniqueandnon‐

overlappingexpressionpatternsofdifferentTGFβligandsduringmulti‐tissue

regeneration.

iii

Acknowledgements

Firstofall,Iwouldliketothatmysupervisors,Dr.MatthewVickaryousandDr.

AliciaViloria‐Petitfortheopportunity,guidanceandsupportthroughoutmydegree.

IwouldalsoliketothankDr.BrendaCoomberforbeingamemberofmyadvisory

committeeaswellasforprovidingexcellentfeedback,guidanceandsupport

throughoutmytimeinthelaboratory.Thethreeofyouhaveprovidedmewiththe

skillsandinsightIneedtobeabletocontributetotheworldofbiomedicalsciences.

Secondly,IwouldliketothankallthemembersoftheCoomber,Vickaryousand

Viloria‐Petitlabsfortheirfriendship,supportandguidance:AmyRichard,Geordon

Avery‐Cooper,SonjaZours,MeghanDoher,PavelNeogi,MahmoudYoussef,Divya

Karsanji,DenisLam,PatriciaHuetherAmandaBarber,NelsonHo,NathanFarias,

LeanneDelaney,KayaSkowronski,JenThompson,JodiMorrison,MaiJarad,

SamanthaPayne,EmilyGilbert,JasonVieraandHannaPeacock.

IwouldliketoespeciallypointouttheextensivetechnicalsupportandtrainingI

receivedfromGeordonAvery‐Cooper,MonicaAntenos,PatriciaHuether,Amanda

Barber,NelsonHo,EmilyGilbertandJodiMorrison.

Finally,Iwouldliketothankmyfriendsandfamilyforsupportingmeandkeeping

megrounded.SpecialthanksgoesouttoAlexandraBaker,AlisonGilbert,Emily

GilbertandDr.RalphGilbert.

iv

IwouldliketodedicatethisthesistoallofthegreatscientistsIhavelookedupto

throughoutmydegreethathaveinspiredmetostrivetoproducemeaningful,

accurate,conciseandcreativeworkthatbenefitsthefieldofknowledgeaswellas

hasthepotentialtochangelives.

v

DeclarationofWorkPerformed

Ideclarethatwiththeexceptionoftheitemsindicatedbelow,allworkreportedin

thisthesiswasperformedbyme.

MembersoftheVickaryouslabpreparedalloftheslidesusedforimmunostaining

(embeddingandsectioning)stainingwasperformedbyRichardW.D.Gilbert.

vi

TableofContents

ABSTRACT II

ACKNOWLEDGEMENTS III

DECLARATIONOFWORKPERFORMED V

LISTOFTABLES VIII

LISTOFFIGURES IX

LISTOFABBREVIATIONS X

INTRODUCTION 1

REVIEWOFLITERATURE 5TRANSFORMINGGROWTHFACTORBETA(TGFβ)SUPER­FAMILY: 5TGFβLIGANDS: 6TGFβACTIVATION 6TGFβLIGAND‐RECEPTORINTERACTIONS: 7ACTIVINLIGANDS 9ACTIVINRECEPTORBINDING 9CANONICALSIGNALLING 11NON‐CANONICALSIGNALING 13TGFβ ISOFORMSINPHYSIOLOGYANDPATHOLOGY. 13TGFβANDACTIVINKNOCKOUTANIMALS 14SMADISOFORMSPECIFICEFFECTS 14CUTANEOUSWOUNDHEALINGANDTGFβ/ACTIVINISOFORMS 15MODELSOFMULTI­TISSUEREGENERATION 17TGFβ/ACTIVINANDMULTI­TISSUEREGENERATION 20TGFβSIGNALINGDURINGAXOLOTLLIMBREGENERATION 20TGFβSIGNALINGDURINGXENOPUSTAILREGENERATION 21TGFβSIGNALINGDURINGZEBRAFISHFINREGENERATION 22LIMITATIONSANDUNANSWEREDQUESTIONS 23EPITHELIALTOMESENCHYMALTRANSITION 25EMTINFIBROSIS 26EMTINCANCER 27EMTINREGENERATION 28THELEOPARDGECKO(EUBLEPHARISMACULARIUS)MODELOFSCARFREEMULTI­TISSUEAPPENDAGEREGENERATION; 29POSTAUTOTOMYMORPHOLOGY:WOUNDHEALING:STAGESI‐III 31POSTAUTOTOMYMORPHOLOGY:REGENERATION:STAGESIV‐VII 32

RATIONALE 34

MATERIALANDMETHODS 36

vii

ANIMALCAREANDHANDLING 36TAILCOLLECTIONANDTISSUESEGMENTATION 36WESTERNBLOTANALYSIS 37IMMUNOFLUORESCENCEANDIMMUNOHISTOCHEMISTRY 38IDENTIFICATIONOFEUBLEPHARISMACULARIUSCDNASEQUENCESUSINGDEGENERATIVEPCR 39QRT‐PCRANALYSIS 40

RESULTS 41TGFβ/ACTIVINSIGNALINGISACTIVEINTHEREGENERATEBLASTEMA. 41PHOSPHORYLATED‐SMAD2ANDPCNAEXPRESSIONINORIGINALANDREGENERATEEPIDERMIS 43PHOSPHORYLATED‐SMAD2ISUPREGULATEDINREGENERATINGTAILS. 44NON‐OVERLAPPINGTGFβ1ANDPSMAD2EXPRESSIONINEARLYTAILREGENERATION. 46IDENTIFICATIONOFNOVELTGFβ/ACTIVINTARGETGENESUPREGULATEDINREGENERATETISSUES. 48

DISCUSSION 57THETGFβ/ACTIVINSIGNALINGPATHWAYISACTIVATEDTHROUGHOUTTHETAILREGENERATIONPROGRAM. 57ACTIVIN,ANDNOTTGFβISUPREGULATED. 58EMTTRANSCRIPTIONFACTORSAREUPREGULATEDDURINGTAILREGENERATION. 61

SUMMARYANDCONCLUSIONS 64

SIGNIFICANCEANDFUTUREDIRECTIONS 67

REFERENCES 70

APPENDIXI–PREPARATIONOFMATERIALS 95

APPENDIXII–CHEMICALLISTANDSUPPLIERS 97

APPENDIXIII–PRIMERSANDCLONEDSEQUENCEANALYSIS 98

APPENDIXIV–CLONEDSEQUENCES 100

viii

ListofTablesTable

1. PrimersusedinqRT‐PCR

p.96

2. PrimersusedtocDNAidentification p.97

3. cDNAsequenceanalysis p.97

ix

ListofFiguresFigure

1. PCNAandpSMADimmunofluorescenceofregenerateblastema p.52

2. PCNAandpSMADimmunofluorescenceofregenerateepidermis

p.53

3. pSMAD2,SMAD2,TGFβ1westernblotofsegmentedgeckotails

p.54

4. TGFβ1immunohistochemistrythroughoutregeneration

p.55

5. Non‐overlappingpSMAD2andTGFβ1expressionpatterns

p.56

6. TGFβ/activinligandgeneexpression p.57

7. EMTtranscriptionfactorgeneexpression p.58

x

ListofAbbreviationsALK Activin‐likeKinaseAPS AmmoniumPersulfateBMP BoneMorphogenicProteinBSA BovineSerumAlbuminDAPI 4’,6’‐Diamidino‐2phenylindoleDTT DithiothreitolECM ExtracellularMatrixEMT EpithelialtoMesenchymalTransitionGDF GrowthandDifferentiationFactorHRP HorseradishPeroxidaseIF ImmunofluoresenceIHC ImmunohistochemistryLAP LatencyAssociatedPeptideLLC LargeLatencyComplexLTBP LatentTGFβBindingProteinMRL MurphyRothsLargePCR PolymeraseChainReactionPBS Phosphate‐bufferedSalinePMSF PhenylmethylsulfonylFluoridePVDF PolyvinylidineFluorideRCF RelativeCentrifugalForceSDS SodiumDodecylSulfateSLC SmallLatencyComplexSMAD Small Body and Mothers Against Decapentaplegic HomologpSMAD PhosphorylatedSMADTβR TransformingGrowthFactorβReceptorTBS Tris‐bufferedSalineTGFβ TransformingGrowthFactorBetaTEMED Tetramethylethylenediamine

1

Introduction

Amongamniotes,themostcommonresponsetotraumaticinjuryistherapid

formationoffibrotictissue.Althoughfibrotic(orscar)tissueisoftencapableof

stabilizingtheinjuryandre‐establishingtissuehomeostasis,itisadysfunctional

andstructurallydissimilarsubstitute(Martin,1997;FergusonandO’Kane,2004;

Gurtneretal.,2008).Incontrast,someamniotes,alongwithvariousurodeles

(newts,axolotls)andteleosts(zebrafish),arecapableofscar‐freewoundhealing

(TanakaandReddien,2011).Significantly,scar‐freewoundhealingisgenerally

followedbytissueregenerationand,inmanyinstances,therestorationoffunction.

Todate,thebest‐knownexamplesofscar‐freewoundhealingandregenerationin

amniotesinclude:earlyembryos,(WhitbyandFerguson,1991;reviewedby

FergusonandO’Kane,2004);thedigittipsofneonateandadultmice,(Hanetal.,

2008;Fernandoetal.,2011;Rinkevichetal.,2011);andthetailofmanylizard

species(BellairsandBryant,1985;Alibardi,2010;Delormeetal.,2012).Lizardsin

particularprovideaninterestingexampleinthatscar‐freewoundhealingand

regenerationistypicallyassociatedwithcaudalautotomy,ananti‐predation

strategywherebythetailisself‐detached.Taillossyieldsalargecross‐sectional

wound,withthespinalcord,skeletalmuscle,dermisandtailvertebraeallexposed

(BellairsandBryant,1985;McLeanandVickaryous,2011).Spontaneously,this

woundisrapidlysealedwithouttheformationoffibrotictissue,andareplacement

tailissoondeveloped(Alibardi,2010;McLeanandVickaryous,2011;Delormeetal.,

2012).Thereplacementtailarisesfromablastema,anaccumulationofhighly

proliferative,mesenchymal‐likecellsthatrapidlypopulatethesiteoftailloss

2

(McLeanandVickaryous,2011).Whereastheendogenoussourceofblastemalcells

inlizardsremainsuncertain,evidencefromthestudyofvariousotherscar‐free

woundhealingandregeneratingspecies,includingurodeles(Kragletal.,2009)and

mice(digittips:Rinkevichetal.,2011)stronglysupportsdedifferentiationof

lineage‐restrictedprogenitorcellsandnotpluripotentstempopulations.

Anemergingmodelforthestudyofscar‐freewoundhealingandregenerationin

lizardsistheleopardgecko,Eublepharismacularius(Whimster,1978;McLeanand

Vickaryous,2011;Delormeetal.,2012).Previousworkintheleopardgeckohas

establishedthatwoundhealingandregenerationarehighlyconservedprocesses

andthattheentireregenerativeprocesscanbedividedintosevendiscrete

morphologicalstages(McLeanandVickaryous,2011).StagesItoIIIprimarily

involvewoundhealing,includingwoundsitecontraction,theformationofan

exudateclot,re‐epithelializationofthewoundsite,andtheinitiationofblastema

formation.StagesIVtoVIIinvolvetissueoutgrowthanddifferentiation,including

angiogenesis,axonogenesis,spinalcordoutgrowth,andmuscleandskeletal

development(McLeanandVickaryous,2011).Althoughthemolecularregulationof

theseeventsremainspoorlyunderstood,evidencefromvariousotherregeneration‐

competentmodelssupportstheinvolvementofvariouscytokines(Stoick‐Cooperet

al.,2007;StocumandCameron,2011)

Thecommoncanonicalsignalingpathwaysharedbytransforminggrowthfactor

beta(TGFβ)andactivinligands,referredtoastheTGFβ/activinpathway(Ogunjimi

3

etal.,2012),hasbeenshowntobeessentialfortheregenerativeresponseinother

modelsofmulti‐tissueregeneration(Jazwinskaetal.,2007;Levesqueetal.,2007;

HoandWhitman,2008).TGFβandactivinaremembersoftheTGFβsuperfamilyof

ligandswhichsignalthroughspecificsetsofserine/threoninekinasereceptors

(MoustakasandHeldin,2009).Uponligandbinding,TGFβ/activinreceptors

becomeactivatedandsubsequentlyrecruitandphosphorylatespecificsignaling

intermediatesknownasreceptorregulatedSMADs(R‐SMADs)(WuandHill,2009).

TGFβandactivincausethephosphorylationofaparticularsubsetofR‐SMADs,

SMAD2andSMAD3(RossandHill.,2008).ThesephosphorylatedSMADsthenform

acomplexwithacommonmediator,SMAD4andsubsequentlytranslocatetothe

nucleusanddriveavarietyofgeneexpressionprograms(RossandHill,2008).The

previousstudiesexaminingTGFβ/activinsignalinginregenerationhaveshownthat

whenthekinasedomainsoftheTGFβandactivinreceptorsarepan‐inhibitedinthe

regeneratingappendagethroughtheuseofsmallmoleculeinhibitors,multi‐tissue

regenerationisblocked(Inmanetal.,2002;DaCostaByfieldetal.,2004).

Additionally,whentheactivinarmoftheTGFβ/activinsignalingpathwaywas

silencedinregeneratingzebrafishtailfinsusingknockdownmorpholinos,

regenerationwaspartiallyblocked(50‐80%reductioninregeneration)(Jazwinska

etal.,2007).Takentogether,theseresultssuggestthatvariousTGFβ/activin

ligands(suchasTGFβ1‐3,and/oractivinβA/ββ)areplayingvital,ligandspecific

rolesinthemultitissueregenerativeprocess.

Despitethesepreviousstudies,theroleofTGFβ/activinsignalingduringmulti‐

4

tissueregenerationremainspoorlyunderstood.Inparticular,thespecific

expressionpatternsofvariousTGFβ/activinligandshavenotbeenextensively

characterizedanditislikelythatdifferentligandsareplayingdifferingrolesat

differenttimesthroughoutregeneration.Forexample,previousworkusingthe

leopardgeckoobservedexclusiveTGFβ3expressioninchondrocytesandfibroblasts

onlyatthelaterstagesofregeneration(VIandVII)(Delormeetal.,2012).These

observations,combinedwiththoseofpreviousstudiesinzebrafish(Jazwinskaetal,

2007),axolotl(Levesqueetal.,2007)andXenopus(HoandWhitman,2008)

suggestingvariableTGFβligandexpressionthroughoutregenerationsuggeststhat

differentialanddynamicexpressionofindividualTGFβ/activinligandsmediate

uniquecellulareventsduringregeneration.

WiththisinmindwesoughttofurtherinvestigatetheTGFβ/activinsignaling

pathwayatmultiplestagesduringtheleopardgeckoregenerativeprogram(from

stagesIIItoVI).Inparticular,wefocusedonthepro‐fibroticcytokineTGFβ1which

hasbeenassociatedwithscarformationandfibrosisinnumerousphysiologicaland

pathologicalsituations(Bielefeldetal.,2012).Wealsosetouttoidentifyingnovel,

TGFβ/activinmediatedtranscriptionaltargetsthatcouldbecontributingtothe

regenerativephenotypeseeninlizards.Inparticular,wefocusedonwell‐

documentedmediatorsoftheTGFβ‐inducedepithelialtomesenchymaltransition

(EMT),aphenomenonofcellularplasticityinvolvedinthereactivationofstemcell

featuresinepithelialcellsthathasyettobeexaminedinthecontextofmulti‐tissue

regeneration(LimandThiery,2012)

5

ReviewofLiterature

TransformingGrowthFactorBeta(TGFβ)Super­family:

Inhumans,theTGFβfamilyconsistsof33members,mostofwhicharedimeric,

secretedpolypeptides(MoustakasandHeldin,2009).Inadditiontothethree

prototypicTGFβproteinisoforms(TGFβ1,TGFβ2andTGFβ3),thefamilyalso

includestheactivins,bonemorphogenicproteins(BMPs),growthand

differentiationfactors(GDFs),myostatinandnodal(reviewedbyDerynckand

Miyazono,2008).TheTGFβfamilyisconservedthroughoutmetazoanevolution,

withligandsbeingfoundinbothvertebratesandinvertebrates(Hinck,2012;

OshimoriandFuchs,2012).Atthecellularlevel,TGFβfamilymembersregulate

growth,differentiation,adhesion,migrationanddeathinacontext‐dependent,dose‐

dependentandcelltypespecificmanner(MoustakasandHeldin,2009).TGFβ

familymembershavealsobeenimplicatedinavarietyofphysiologicaland

pathologicalprocessesincludingdevelopment(reviewedbyMoustakasandHeldin,

2009),stemcellmaintenance(reviewedbyBeyeretal.,2012;OshimoriandFuchs,

2012),fibrosis,autoimmunedisease,cardiovasculardisease(reviewedbyGordon

andBlobe,2008),andcancer(reviewedbyMassague,2008).Inaddition,ligands

fromtheTGFβsuperfamilyhavedocumentedrolesduringwoundhealingand

regeneration(Jazwinskaetal.,2007;Levesqueetal.,2007;HoandWhitman,2008;

Bielefeldetal.,2012).

6

TGFβLigands:

Amongmammals,theTGFβisoformsTGFβ1,TGFβ2andTGFβ3aremultifunctional

cytokinesthatactinautocrineandparacrinemannerstoregulateadiversearrayof

cellularprocesses(tenDijkeandArthur,2007).Eachoftheseisoformsisencoded

byaseparategene,withthepreproteinsequenceconsistingofasignalpeptide(~30

kDa)andthe“mature”TGFβsequence(~12.5kDa)(MungerandSheppard,2011).

Afterproteolyticprocessingintheendoplasmicreticulum,theTGFβmonomers

formhomodimersthroughdisulfidebonding,resultingintheformationofthe

precursormolecule,pro‐TGFβ(tenDijkeandArthur,2007).Thepro‐TGFβdimeris

thenfurtherprocessed(byfurinconvertase)toyieldasmalllatentcomplex(SLC)

consistingofthematureTGFβproteinandalatencyassociatepeptide(LAP)(Dubois

etal.,1995).TheSLC(matureTGFβproteinplusLAP)thenformsacomplexwith

thelargelatentTGFβbindingprotein(LTBP)toyieldthelargelatencycomplex

(LLC)(Rifkin,2005).OncesecretedtheLLCinteractsthroughitsN‐terminalwith

variousextracellularmatrixcomponentstoallowTGFβtobecomedepositedinthe

ECM(tenDijkeandArthur,2007)

TGFβActivation

InorderforlatentTGFβtobecomeactivated,thematureTGFβdimermustbe

releasedfromtheLLC(TGFβ‐LAP‐LTBP)complexembeddedintheECM.Thiscanbe

achievedthroughavarietyofdifferentmechanismsinvariouscellularcontexts,all

7

ofwhichdirectlytargettheLAP(reviewedbytenDijkeandArthur,2007).For

example,invivoithasbeendemonstratedthatbindingofsecreted

thrombospondin‐1totheLAPdisruptsthenon‐covalentbondsholdingTGFβ,

leadingtoreleaseandactivation(Crawfordetal.,1998).Furthermore,itisknown

thatlatentTGFβcanbeactivatedbyαvβ6andαvβ8integrins,whichbindtheRGD

sequenceintheLAPandcauseaconformationalchange(Sheppard,2005).The

importanceofTGFβactivationisdemonstratedinmicewithengineeredmutations

invariousTGFβactivatorsexhibitingphenotypicrecapitulationsofmicedeficientin

TGFβsignalingcomponents(Yangetal.,2007;summarizedbytenDijkeandArthur,

2007).Asanexample,ithasbeendemonstratedthatmicewithanengineered

mutationintheRGDsequenceoftheLAPrecapitulatethemainfeaturesofTGFβ1

nullmice(Yangetal.,2007).

TGFβLigand­ReceptorInteractions:

OncematureTGFβpeptidesarereleasedfromtheLAP,theseligandsevoketheir

cellulareffectsintargetcellsbybindingtotransmembrane,dualspecificity

receptors(strongserine/threonineactivityandweaktyrosinekinase

activity)(MoustakasandHeldin,2009).TGFβreceptorscanbedividedintothree

classes:typeI,typeIIandtypeIII(co‐receptor)(TβRI,TβRIIandTβRIII,

respectively).SeventypeI,fivetypeIIandtwotypeIIIreceptorshavebeen

identifiedinhumans(ShiandMassague,2003,Wrana,2008)TypeIandtypeII

receptorshaveshortextracellularcysteine‐richdomainsthataresubjectto

8

glycosylation,asinglepasstransmembranedomain,andanintracellulardomain

consistingmainlyofthekinasecatalyticmotif(Wranaetal.,2008;Kangetal.,2009).

TGFβligands(TGFβ1‐3)primarilybindtoTβRIIandTβRI,alsoknownasactivin

receptor‐likekinase5(ALK‐5).Toactivatecellsignaling,theTGFβligandfirstbinds

totheconstitutivelyactiveTβRII,whichisthenbroughtintocloseproximitywith

TβRI(Wranaetal,1994).ThiscloseinteractionallowsTβRIItophosphorylatethe

shortGSdomain(TTSGSGSG)locatedatthejuxtamembraneregionofTβRI.

PhosphorylationresultsinaconformationalchangetoTβRI,leadingtoreceptor

activation(Souchelnytskyietal.,1996;Kangetal.,2009).Evidencesuggeststhat

bothTβRIandTβRIIexistashomodimersonthecellmembrane(TypeI‐TypeI,

TypeII‐TypeII)butcomplexintoheterotetramersinthepresenceofaligand

(Yamashitaetal.,1994).Ascurrentlyunderstood,allTGFβsignalingrequiresboth

TβRIandTβRII(Wranaetal.,2008).Insupportofthis,ithasbeenshownthanTGFβ

ligandsonlybindtoTβRIIwithhighaffinity,thusmakingreceptorheterotetramer

complexformationcriticalforTβRIactivation(Wranaetal.,2008).Onceactivated,

thereceptorcomplexinitiatesanintracellularcascadethatevokestheactivationof

canonicalandnon‐canonicalsignalingpathways(discussedbelow;reviewedby

MoustakasandHeldin,2005,2009).Addingtothecomplexityofthisprocess,some

ligand‐receptorinteractionsrequireaTβRIIIco‐receptor.Forexample,TβRIIis

unabletobindTGFβ2withoutaco‐receptor,suchasbetaglycan(DeCrescenzoetal.,

2006).

9

ActivinLigands

TheTGFβsuperfamilyalsoincludesactivins,andthestructurallyandevolutionarily

relatedinhibinsthat,similartoTGFβligands,areknowntohavediversefunctions

throughoutthebody,inparticularduringembryonicdevelopmentandwound

healing(WiaterandVale,2008).Activinsandinhibinsarebothdisulfidelinked

dimersconsistingofvariousinhibin‐αandinhibin‐βpolypeptidesubunits.In

humansthereisasingleinhibin‐αpolypeptideandfourinhibinβpolypeptides(βA,

βB,βC,βD)allencodedbydifferentgenes(Vittetal,2001).Thematureinhibin

dimers(inhibin‐A,B)existasheterodimersofaninhibin‐αsubunitcombinedwith

aninhibin‐βsubunit(βAorβB)(WiaterandVale,2008).Thematureactivindimers

(ActivinA,B,AB)existashomodimersofvariousinhibinβsubunits(Burgeretal.,

1988;Valeetal.,1988).SimilartoTGFβ,activinandinhibinsubunitsareinitially

synthesizedaslargeprecursorproteinsthatrequireproteolyticprocessingfor

properfoldingandsecretion.Briefly,theprodomainsoftheinhibinsubunitsare

cleavedafterdimerassemblytoyieldthematureactivinorinhibinpolypeptide

(WernerandVale,2008).UnlikeTGFβligands,thepro‐domainofactivinandinhibin

doesnotstayincontactwiththematurepeptideandhasnoapparentbiological

effect(WiaterandVale,2008).

ActivinReceptorBinding

AsforTGFβligands,activinsinduceresponsesintargetcellsbybindingtotypeII

10

andtypeIreceptors.MammalshavetwoactivintypeIIreceptors,ActRIIAand

ActRIIB,andoneactivintypeIreceptor,ActRIB(alsoknownasAlk‐4)(Carcamoet

al.,1994;Attisanoetal.,1996).Themolecularinteractionsbetweenthevarious

activinligandsandtheirreceptorsaresimilartothoseofTGFβligands.Forexample,

similartotheTGFβligandreceptorinteractions,activininteractsfirstwithActRIIA

orBandthenActRIB,causingtheactivationofthetypeIkinasedomains(Wiater

andVale,2008).Itissuggestedthatactivin‐mediatedactivationinvolvesthesame

canonicalandnon‐canonicalpathwaysobservedfollowingactivationwithTGFβ

ligands,howeverthisislikelyanoversimplificationanddownstreamsignaling

differencesarehighlyprobable(MoustakasandHeldin,2005;WiaterandVale,

2008;MoustakasandHeldin,2009).

Inhibinligandsareknowntoantagonizetheactionsofactivinsparticularilyamong

tissuesofthereproductiveaxis(Harrisonetal.,2005).Inhibinantagonismofactivin

signalingresultsfromcompetitionforActRIIAorB,mediatedthroughreceptor

bindingtothesharedinhibinβsubunit(βAorββ)(Grayetal.,2000).Itisworth

notinghowever,thatinhibinaffinityforActRIIBisten‐timeslowerthanactivin,and

thatinsometissuesinhibincannotreadilyantagonizeactivin(Lewisetal.,2000).It

hasbeendemonstratedthatthetypeIIITGFβco‐receptorbetaglycancanactasaco‐

receptorforinhibin,leadingtoathirty‐foldincreaseininhibinbindingaffinityatthe

ActRIIB(Lewisetal.,2000).Interestingly,betaglycanisdifferentiallyexpressedin

tissuesthroughoutthebody,andintherat,betaglycanmRNAisexpressedinthe

brain,pituitaryglandandgonads,pointingtotissuespecficrolesforinhibin

11

(MacConelletal.,2002).

CanonicalSignalling

ThecanonicalTGFβ/activinsignalingpathwayismediatedthroughcytoplasmic

proteinsknowasSMADs(Smallbodyandmothersagainstdecapentaplegic

homolog)(reviewedbyFengandDerynck,2005).Invertebratesthereareeight

membersoftheSMADfamily,SMADs1‐8.EachSMADproteinconsistsofthree

domains:(1)anN‐terminalMad‐homology(MH)1domainthatcaninteractwith

otherproteinsandcontainsthenuclearlocalizationsignalaswellasDNAbinding

elements;(2)amiddlelinkerdomainthatinteractswithubiquitinligases;and(3)a

C‐terminalMH2domainthatbindstypeIreceptors,interactswithotherproteins,

andmediatesSMADhomo‐andhetero‐oligomerization(FengandDerynck,2005;

MoustakasandHeldin,2009).SMADsarefurthercategorizedintothreeclasses

dependingontheirstructureandfunction.ReceptoractivatedorR‐SMADs(SMADS

1‐3,5,8)interactwithactivatedtypeIreceptorsresultinginphosphorylationoftheir

conservedSSXSmotif,locatedattheproteincarboxy‐terminus(C‐terminus)(Feng

andDerynck,2005).UponC‐terminalphosphorylationthecommon‐mediator

SMAD,Co‐SMAD(SMAD4),interactswithactivatedR‐SMADcomplexestoassist

theirnucleartranslocation.InhibitoryorI‐SMADs(SMADs6,7)interactwith

activatedtypeIreceptorsandinactivatethereceptorcomplexwiththehelpofthe

E3ubiquitinligaseSmurf1/2(FengandDerynck,2005;MoustakasandHeldin,

2009).

12

ThefiveknownR‐SMADs(SMADs1‐3,5,8)arephosphorylatedinresponseto

differentTGFβfamilyligandsthatbindvariousTGFβreceptorcombinations.In

mostcasesTGFβ,activin,myostatinandnodalligandscauseC‐terminal

phosphorylationofSMAD2andSMAD3,whereasBMPsandGDFscausetheC‐

terminalphosphorylationofSMAD1,SMAD5andSMAD8(RossandHill,2007).

However,ithasbeenreportedthatTGFβ1‐3cancauseC‐terminalphosphorylation

ofSMAD1/5/8throughacell‐typespecifictypeIreceptor(ALK‐1)foundon

endothelialcells(Goumansetal.,2002).Uponreceptor‐inducedC‐terminal

phosphorylation,R‐SMADsinteractwithSMAD4(theco‐SMAD),formingactivated

SMADcomplexes.SMAD4isessentialformostSMADtranscriptionalcomplex

formationaswellasnucleartranslocation,andisnotligandrestricted(i.e.,SMAD4

canformcomplexeswithallR‐SMADS).InresponsetoTGFβ/activinsignaling,

activatedSMADcomplexesmayformheterodimers(SMAD2or3‐SMAD4)or

heterotrimers(SMAD2‐SMAD3‐SMAD4),dependingonthetargetgene.These

complexesaccumulateinthenucleusandcanregulategeneexpressionboth

positivelyandnegativelyofteninassociationwithothertranscriptionfactors(Feng

andDerynck,2005;RossandHill,2007).AddingtothecomplexityofSMAD

signalling,eachoftheR‐SMADproteinscontainsnumeroussitesforpost‐

translationalmodification,includingmultiplephosphorylationsites,sumolation

sites,aswellasacetylationsites.Thesemodificationscancausenumerouseffectson

SMADsignalling,furtherreviewedby(MoustakasandHeldin,2009,Xuetal.,2012)

13

Non­CanonicalSignaling

WhereasTGFβ/activinligandsprimarilysignalthroughthecanonicalSMAD

pathway,numerousnon‐canonical/non‐SMADsignalingpathwayshavealsobeen

demonstratedtobeactivatedbyTGFβligands.TheseincludetheRas/MAPK/Erk

pathway,thePI3K/Aktpathway,theJNK/SAPKpathway,thep38MAPKpathway,

andthePar6‐Polaritypathway.Non‐canonicalpathwaysplaydiverserolesin

modulatingSMADmediatedandnon‐SMADmediatedsignalingevents,andcanbe

activateddirectlyorindirectlybytheTGFβreceptorcomplex.Theimportanceof

non‐canonicalsignalingindeterminingthefunctionaloutcomeofTGFβ/activin

signalinghasbeenpreviouslydemonstrated(Ozdamaretal.,2005;Leeetal.,2007;

Sorrentinoetal.,2008;Yamashitaetal.,2008).Thisthesiswillfocusoncanonical

signaling,givenitswell‐documentedroleintissueregeneration(Jazwinskaetal.,

2007;Levesqueetal.,2007;HoandWhitman,2008).Arecentreview(Zhangetal.,

2009)presentsanextensivediscussiononnon‐canonicalsignalinganditsrolein

TGFβ’smultiplecellulareffects.

TGFβ isoformsinphysiologyandpathology.

Despitesharing71‐76%sequenceidentityandsignalingthroughthesamecanonical

SMADintermediates,agrowingbodyofevidencesuggeststhatthethreeTGFβ

isoformsaswellasthevariousactivinisoformscausedifferenttranscriptional

effects(FergusonandO’Kane,2004,Nawshadetal.,2004).Inparticular,ithasbeen

14

observedthatTGFβ1/2/3andactivinknockoutmicealldemonstratedistinct,

generallynon‐overlappingphenotypes.

TGFβandactivinknockoutanimals

TargeteddisruptionofTGFβ1geneleadstohematopoeticandvasculogenicdefects

thatresultinthedeathofnearly50%ofnullembryosby10daysofgestation

(Dicksonetal.,1995).Theremainingembryoseventuallydieofwastingsyndome

andmulti‐organfailureasaresultofexcessivemultifocalinflammatorydisease

(Shulletal.,1992).TGFβ2knockoutmiceexhibitperinatalmortalityasaresultof

multipledevelopmentalabnormalitiesaffectingthecardiopulmonary,urogenital,

visual,auditory,neuralandskeletalsystems(Sanfordetal.,1997).Micelacking

TGFβ3exhibitcleftpalateanddieimmediatelyafterbirthduetoaninabilityto

suckleeffectively(Proetzeletal.,1995;Kaartinenetal.,1995;Lavertyetal.,2009).

Duetooverlapbeweenacitvinandinhibin(mRNAtranscriptsthatencodeactivin

monomersalsomakeupcomponentsoftheinhibinsystem),accurateinterpretation

ofactivinknockoutmiceremainsproblematic(ValeandWerner,2008).Itisworth

notinghowever,thatactivin­βAknockoutmicedemonstratedelayedvibrissae

follicleformationand(similartoTGFβ3knockouts)cleftpalateabnormalities

(Matzuketal.,1995;McDowalletal.,2008).

SMADisoformspecificeffects

15

SimilartothevariousisoformsofTGFβandactivin,SMAD2andSMAD3appearto

havedifferentrolesduringdevelopmentaswell.SMAD2and3arehighlyconserved

attheproteinlevel,with83.9%homology(Petersenetal.,2010).However,thereis

animportantstructuraldifferenceintheMH1domain,whereSMAD2containstwo

shortpeptideinsertsthatimposestericconstraintsthatpreventitfrombinding

DNA,whereasSMAD3retainsDNAbindingcapacity(Dennleretal.,1999).In

additiontostructuraldifferences,micedeficientinSMAD2areembryoniclethal

whereasSMAD3deficientmiceareviable(Weinsteinetal.,1998;Ashcroftetal.,

1999;Yangetal.,1999).Interestingly,keratinocytespecificSMAD3knockoutyields

micethatareresistanttocarcinogeninducedskincancerwhilekeratinocytespecific

SMAD2knockoutleadstoacceleratedformationandmalignantprogressionof

carcinogeninducedtumorssuggestingdistincttranscriptionaleffects(Hootetal.,

2008).

CutaneousWoundHealingandTGFβ/activinisoforms

OneofthebestexampleshighlightingthecomplexityofthevariousTGFβ/activin

isoformsinthedeterminationofphysiologicalandpathologicaloutcomesis

cutaneouswoundhealing.Cutaneouswoundhealingisahighlydynamicprocess

regulatedthroughnumerouscellularandmolecularinteractions(RienkeandSorg,

2012).Inadultmammalscutaneouswoundrepairinvolvessuccessivesstagesof

hemostasis,inflammation,granulation,andsubsequentreepithelizationresultingin

theformationofscartissue(RienkeandSorg,2012).Maturemammalianscartissue

16

ischaracterizedasavascularandacellularconsistingofmainlydisorganizedECM

(RieinkeandSong,2012).Comparedtopost‐natalmammalianwoundhealing,

mammalianfetusesretaintheremarkableabilitytoundergoscar‐freewound

healing(Rowlatt,1979;FergusonandO’Kane,1996;Colwelletal.,2006).In

responsetonumeroustissueinjuries,fetaldermisandepidermisregenerates

nondisruptedECManddermalappendagesindistinguishablefromorignaltissue

(Coolenetal.,2010).Thisscar‐freewoundhealingcapacityhasbeenattributedto

numerousfactorsincludingECMdifferences,alteredinflammatoryresponse,

differentialgene/proteinexpressionaswellasvariedstemcellfunction(Reinkeand

Song,2010).Interestingly,differentTGFβligandshavebeenshowntobe

differentiallyexpressedduringfetalwoundhealing.Notably,verylowlevelsof

TGFβ1andTGFβ2,andhighlevelsofTGFβ3havebeenreported(Whitbyand

Ferguson,1991;FergusonandO’Kane,1996).Furthermore,whenexogenousTGFβ1

isappliedtofetalwounds,scarformationreminiscentofadultwoundhealingis

observed(LinandAdzick,1996).Buildingonthis,ithasbeendemonstratedthatin

adultcutaneouswoundsreductioninTGFβ1andTGFβ2orexogenousapplicationof

TGFβ3canreducescarformation(Shahetal.,1995;FergusonandO’Kane,1996).

TGFβ1hasbeenextensivelyreportedtobepro‐fibroticandpro‐inflammatory,

(reviewedbyBiernackaetal.,2011)andTGFβ2hasalsobeendemonstratedto

contributetofibrosispromotioninnumeroussituationsaswell(Cordeiroetal.,

1999;Hilletal.,2001).ConflictingevidenceexistsinregardstotheroleofTGFβ3in

scarformationasithasbeenreportedthatTGFβ3isbothpro,andantifibrotic

(Lavertyetal.,2009).Inregardstothis,arecentclinicaltrialinvolvingcutaneous

17

applicationofTGFβ3forscarreductionfailedtomeetexpectedoutcomes(Wuetal.,

1997;Occlestonetal.,2011).Activinligandshavealsobeenshowntoplayarolein

cutaneouswoundhealing(WernerandAlzheimer,2006).Followingwounding,

bothmiceandhumansdemonstrateupregulationofactivinA,howeverexpression

levelshavenotbeenexaminedinmammalianfetalwoundhealing.

Thesevariousobservationsinbothmammalianscar‐freefetalwoundsandscar

formingadultwoundsalludetocomplexandpoorlyunderstoodisoformspecific

effectsofTGFβ/activinligandsinthewoundhealingprocess.Therefore,establishing

thespecificrolesofdifferentTGFβ/activinisoformsduringscar‐freewoundhealing

andmulti‐tissueregenerationinadultamniotesisessentialtoourunderstandingof

thedifferencesintheseprogrammesinadulthumansandholdsgreatpromiseinthe

fieldofregenerativemedicine.Interestingly,someorganismsretaintheabilityto

healwoundswithoutscarformationintoadultlife.Thenextsectionspresentan

overviewonourcurrentunderstandingofpost‐natalscar‐free,multi‐tissue

regenerationandtheevidenceoftheroleofTGFβ/activinsignalingintheprocess.

ModelsofMulti­TissueRegeneration

ThereexistsastrikingdiversityofregenerativepotentialthroughoutMetazoa.

Variousinvertebrates,suchasplanarians,areabletoregeneratevirtuallyevery

portionofthebody(Sanchez‐Alvarado,2006).Amongvertebrates,theregenerative

potentialisconsiderablymoremodest.However,variousspeciesarecapableof

replacinglostordamagedappendagesandorgans.Amongthemostcommonly

18

recognizedregeneration‐competentspeciesare:variousteleostfish(e.g.,zebrafish),

whichcanregenerateportionsoftheirheart,jaws,spinalcordsandtailfins

followingvariousinsults(Poss,2007;Taletal.,2010);Xenopustadpoles,thatcan

regeneratetheirtails,limbsandretina(TsengandLevin,2008);andurodele

amphibians(newtsandaxolotls)thatareabletoregeneratetheirtails,limbs,spinal

cords,andinsomecasesheartandretina(Brockes,1984;Tsonis,1996;Songand

Stocum,2010).Amongstamniotes,someofthemostimpressiveexamplesofmulti‐

tissueappendageregenerationcomefromlizards.Representativemembersofmost

majorlineages,includingpolychrotids(e.g.,Anoliscarolinensis)andgekkotans(e.g.,

Eublepharismacularius)areabletoregeneratetheirtailfollowinglossofthe

appendage(BellairsandBryant,1985;McLeanandVickaryous,2011).Another

notableexampleofamnioteregenerationisdigittipregeneration,asdemonstrated

inrodentsandprimates(Illingworth,1974;VidalandDickson,1993;Rajnochetal.,

2003;Muneokaetal;2008).Furthermore,severalspeciesofmicepossessenhanced

regenerativecapacityincludingspinymice(Acomys)(Seifertetal.,2012),MRL

(MurphyRothsLarge)mice(Herber‐Katz,1999;LeferovichandHerber‐Katz,2002),

andmicewithapointmutationinTGFβR1(Liuetal.,2010).

Mostexamplesofmulti‐tissueregenerationinvertebrates,withthepossible

exceptionofmouseandprimatedigittips(Muneokaetal.,2008),beginwiththe

formationofamassofmesenchymal‐likecellsatthewoundsite(Wallace,1981;

Tsonis,1996;Tamuraetal.,2010).Thiscellularaggregation,commonlyreferredto

asablastema,ismorphologicallysimilartoanembryoniclimbbud(Hanetal.,

19

2005).Blastema‐mediatedorepimorphicregenerationischaracterizedbyan

aggregationofproliferatingcellsthatgivesrisetoreplacementtissues(Carlson,

2007).Morphologically,theblastemaappearstobecomposedofahomogenous

populationofundifferentiatedcells.However,variousrecentstudieshave

demonstratedthatblastemacellsareactuallyaheterogenouspooloflineage

restrictedprogenitorcells(BrockesandKumar;2002;Kragletal,2009;Rinkevich

etal.,2011;Lehoczkyetal.,2011).Consequently,blastemacellsarenota

pluripotent(orperhapsevenmultipotent)population,butinsteadretainamemory

oftheirgermlayerorigin(axolotls:Kragletal.,2009;mousedigits:Rinkevichetal.,

2011).Detailsofblastemaformationremainpoorlyunderstood,butitispredicted

thateitherreprogrammingeventsoccurringamongstthedifferentlineagerestricted

progenitorcellpopulationsthatmakeuptheblastema,orrapidexpansionoftissue

specificstemcellpopulations,oracombinationofbothprocessesgiverisetothe

blastema(Kragletal.,2009;Rinkevichetal.,2011;Joplingetal.,2011).

Throughouttheprocessofepimorphicregeneration,thewoundsiteanddeveloping

blastemaiscappedbyaspecializedwoundepithelium.Thewoundepitheliumfirst

formsshortlyafterappendageloss,asoriginalepidermalcellssurroundingthe

woundquicklymigrateacrossthesiteofinjury(Stoick‐Cooperetal.,2008).Once

thewoundareaiscovered,thewoundepitheliumbeginstothicken,givingrisetoan

apicalcapcomparablewiththeapicalectodermaridge(AER)observedduringlimb

development(ChristensenandTassava,2000;NacuandTanaka,2011).Thiswound

epitheliumlacksthedistinctivestratifiedappearance,basalkeratinocytepolarity

20

andmaturebasallaminaobservedinpre‐woundingepidermis(Globusetal.,1980;

NeufeldandDay,1996).Matchingitsdistinctiveappearance,thewoundepithelium

demonstratesuniqueproteinandgeneexpressionprofilescomparedtonormal

epithelium(Hanetal.,2005,reviewedbyCampbellandCrews,2008;Campbellet

al.,2011;Monaghanetal.,2012).Numerousreportshaveestablishedthatthe

woundepitheliumisvitalforblastemainductionandproliferation(Thornton,1957;

ChristensenandTassava,2000;NacuandTanaka,2011).

TGFβ/activinandmulti­tissueregeneration

Todate,aspectsofTGFβ/activinsignalinghavebeeninvestigatedinthreedifferent

experimentalmodelsofmulti‐tissueregeneration:limbregenerationinaxolotls

(Levesqueetal.,2007),tailregenerationinXenopus(HoandWhitman,2008)and

finregenerationinzebrafish(Jazwinskaetal.,2007).

TGFβsignalingduringaxolotllimbregeneration

Asforotherexamplesofepimorphicregeneration,axolotllimbregenerationbegins

withaperiodofwoundhealingfollowedbyblastemaoutgrowthandtissue

regeneration.ThroughoutaxolotllimbregenerationTGFβ1mRNAexpressionis

upregulated(Levesqueetal.,2007).Injectingthesmallmoleculeinhibitorof

TGFβ/activinsignaling,SB‐431542,intothelimbstumpdelaystheformationof

woundepithelium,limitsblastemacellproliferationandultimatelyimpedes

21

blastemaformation(Levesqueetal.,2007).

TGFβsignalingduringXenopustailregeneration

AmongXenopustadpoles,allofthetissuesofthetailincludingskin,neuraltissue

andmuscle,canbefullyregeneratedfollowingamputation.Thisregenerative

capacityisrestrictedtotadpolesanddiminishesandstopsduringmetamorphosis

(Slacketal.,2004).Ashasbeendemonstratedformodelsofepimorphic

regeneration,genesactiveduringembryonicdevelopmentreactivateduring

Xenopustadpoletailregeneration(reviewedbyPoss,2010;NacuandTanaka,

2011).Within15minutesoftailamputation,thereisanincreaseinphosphorylated

SMAD2(pSMAD2)immunostaining,suggestingactivationoftheTGFβ/activin

pathway.Interestingly,thismaybetheearliestreportedsignalingeventthatoccurs

inregeneration(HoandWhitman,2008).ThisearlypSMAD2activationisrestricted

toasinglelayerofepidermalcellsneartheamputationsiteatthecutedge.

However,asregenerationproceeds,pSMAD2expressionbecomesmore

widespread,includingmanycellsoftheblastema,epidermis,andoriginal

notochord.UpregulationofTGFβ/activinfamilymembersxTGFβ2(similarto

TGFβ2),xTGFβ5(similartoTGFβ1),xGDF11andxActivin­βAwasobservedusing

semi‐quantitativePCR(HoandWhitman,2008).WhenSMAD2/3phosphorylation

wasinhibitedatthetimeofamputation(usingthesmallmoleculeinhibitorSB‐

431542)boththewoundepidermisandtheblastemafailedtoform(Hoand

Whitman,2008).Furthermore,blockadeofSMAD2/3phosphorylationwithSB‐

22

431542alsoappearedtoinhibitothersignalingpathwaysknowntobeactivated

duringregeneration,includingBMPandErk(HoandWhitman,2008).Theauthors

concludethat,TGFβ/activinsignalingisrequiredfortheformationofthewound

epitheliumandtheestablishmentofblastemathateventuallyconstitutethe

regenerateappendage(HoandWhitman,2008).

TGFβsignalingduringzebrafishfinregeneration

ThemostextensiveinvestigationofTGFβ/activinsignalingduringmulti‐tissue

regenerationcomesfromthestudyofzebrafishfinregeneration(Jazwinskaetal.,

2007).Usingmicroarrayanalysis,theauthorssoughttodeterminewhich

transcriptswereupregulatedatthreedifferentearlytimepointsfollowing

amputation:0hours,6hours(re‐epithelializationcompleted)and24hours(startof

blastemainduction).Themicroarrayscreenrevealedupregulationofactivin­βA

(Jazwinskaetal.,2007).Toconfirmthemicroarraydata,qRT‐PCRwasperformed

examiningalltheTGFβfamilyligandsknowntosignalthroughSMAD2/3(viz.

TGFβ1­3,activin­βAand­βB,andGDF9­11)atthesamethreeearlytimepoints.

Validatingthemicroarraydata,onlytheactivin­βAtranscriptwassignificantly

upregulated,increasing~18timesatsixhourspost‐amputationand15timesat24

hours,whencomparedto0hourstimepoint.Usingin‐situhybridization,activin­βA

expressionwaslocalizedtotheinterraypocketsformedbetweenthewound

epidermisandblastema,andappearedtocontinuethroughoutregeneration

(Jazwinskaetal.,2007).UsingthesmallmoleculeinhibitorSB‐431542theauthors

23

furtherdemonstratedthatSMAD2/3inhibitionresultsinanabnormalwound

epidermisandlackofblastemaformation(asseeninXenopusandaxolotl;Levesque

etal.,2007;HoandWhitman,2008).Tofurtherexaminethesignalingmechanism,

theauthorsusedatemperaturesensitivemutantforfgf20a.Onceheatshocked,

thesefishdonottranscribefgf20aand,asaresult,ablastemafailstoformfollowing

tailamputation(Whiteheadetal.,2005).Activin­βAexpressionwasstillobservedin

theinterraypocketsofthesemutants,suggestingthatactivinupregulationdoesnot

requireFGFsignaling,orblastemaformation(Jazwinskaetal.,2007).Toelucidate

theroleofactivin­βAinregeneration,knockdownmorpholinoswereusedtoablate

thisgene(activin­βA),aswellastheAlk4receptorgene.Theresultwasa50%and

80%reductioninregeneratetailsizerespectivelysuggestingotherligandsmayplay

redundantrolesduringregeneration.

Limitationsandunansweredquestions

PreviousworkhasoftenfocusedontheuseofthesmallmoleculeinhibitorsSB‐

431542andSB‐505124.Itisimportanttonotehowever,thatthesemolecules

inhibitthephosphorylationofallcanonical(SMAD)mediatedTGFβ/activin

signaling(i.e.bothSMAD2and3).SinceSMAD2and3canbephosphorylatedby

eitherTGFβoractivinligands,useoftheseinhibitorsisnotligandspecific.Observed

differencesintheupregulationofvariousTGFβfamilyligandssuggeststhateach

maybeplayinguniqueandnon‐overlappingrolesduringregenerationandpossibly

indifferentspeciesanddifferentregeneratingstructures(Levesqueatal.,2007;

24

Jazwinskaetal.,2007;HoandWhitman,2008).Furthermore,activin­βAandAlk­4

knockdownmorpholinosfailedtocompletelyrecapitulatetheeffectsofSB‐431542

treatment,suggestingotherTGFβligandsbesidesjustactivinareplayingroles

duringregeneration.

Together,thesestudiesdemonstratethatTGFβplaysacriticalroleduring

regenerationbutdonotconclusivelyindicatewhichligandsareresponsiblefor

drivingthelargeinductionofpSMAD2/3.Evidencefromthestudyofzebrafishfin

regenerationsuggeststhatactivin‐AisthemainligandresponsibleforthepSMAD

induction,butdoesnotruleoutotherligands.Thesefindingssuggestthatthe

expressionofvariousTGFβfamilyligandsshouldbefurtherexaminedwithhighly

sensitivetechniquestoclarifythedifferencesseenindifferentmodelsofmulti‐

tissueregeneration.Inparticular,mostworkexaminingTGFβ/activinligand

expressionhasexaminedmRNAlocalization,thusworkexaminingprotein

localizationwouldcomplementthecurrentdata.Furthermore,theextremelyrapid

inductionofTGFβ/activin–SMAD2/3mediatedsignalingseeninXenopusand

zebrafish,combinedwiththelossoffunctionstudies,suggeststhattheseunknown

TGFβ/activinligandcombinationsareplayingimportant,instructiverolesin

dictatingtheinitiationandsuccessofregeneration.Onceagain,theseevidences

emphasizetheneedforabetterunderstandingofthedifferentrolesplayedby

individualTGFβligandsinregeneration.

25

EpithelialtomesenchymaltransitionOnesuchrolethatvariousTGFβ/activinligandscouldbemediatingduringmulti‐

tissueregenerationistheepithelialtomesenchymaltransition(EMT).Theepithelial

tomesenchymaltransitionisanevolutionaryconservedprocessthatoccursduring

development,woundhealing,fibrosisandcancermetastasis.EMTinvolvestheloss

ofepithelialphenotypeandtheacquisitionofamesenchymalphenotype(Nieto,

2011;LimandThiery,2012).Thistransitionismediatedbyasetoftranscription

factors(Namely;Snail1,Snail2,Zeb1,Zeb2andTwist1andTwist2)thatpromote

cellularmotility.EMTtranscriptionfactorsareupregulatedbyavarietyof

extracellularstimuli,includingFGF,WntandTGFβfamilysignalling(Peinadoetal.,

2007;LimandThiery,2012).Importantly,EMTisreversibleandcellscancycle

betweenepithelialandmesenchymalstatesviaEMT,andthereverseprocessMET

(mesenchymaltoepithelialtransition)(LimandThiery,2012).Furthermore,EMT

appearstobecontextdependentandoccurs,muchlikeSMADsignaling,withinthe

frameworkofothersignalingmechanismssuchasfateinductionanddifferentiation

(Nieto,2011;LimandThiery,2012).

Throughoutdevelopment,EMToccursinsuccessivewavesduringboth

morphogenesisandorganogenesisplayingrolesingastrulation,neuralcrest

delamination,cardiaccushionandvalveformationaswellaspalatefusion.

(Nashawdetal.,2004,Nieto,2011;LimandThiery,2012).Intheadult,ithasbeen

proposedthatEMTplaysaroleduringwoundhealingandinsupportofthisithas

recentlybeenshownthatSnail2isupregulatedfollowinginjurytocornealepithelial

26

cells(SavagnerandArnoux,2009;Aomatsuetal.,2012;Leopoldetal.,2012).

DespitethisobservationtheroleoftheEMTinwoundhealingremainspoorly

understoodandrequiresfutherexamination.Currently,thebest‐studiedexamples

ofpostnatalEMTinvolvepathologicalconditionssuchasfibrosisandcancer

metastasis.

EMTinfibrosis

EMThasbeensuggestedtobeapotentialdriverofcardiacandrenalfibrosis.Using

transgeniclineage‐tracingstrategiesithasbeendemonstratedthatupto30%of

activatedfibroblastsinfibroticcardiacandrenaltissueareofanapparent

endothelialorepitheliallineagesuggestinganEMTtransitioncouldunderlytheir

activation(Zeisbergetal.,2007;Zeisbergetal,2008).EMThasalsobeenshownto

driveexperimentallyinducedliverfibrosisandTGFβinducedlungfibrosisin

severalstudies(reviewedby:QuagginandKapus,2011).Despitetheseresults,the

roleofEMT(s)duringfibrosisremainsatopicofconsiderabledebateasacauseand

effectrelationshipremainsdifficulttodetermine(seeQuagginandKapus,2011).

Furthermore,EMThasalsobeensuggestedtoplayaroleinthegenerationofcancer

associatedfibroblasts(CAF)whicharedescribedasactivatedfibroblaststhat

contributetocancerprogressionandmetastasis(KalluriandZeisberg,2006).

27

EMTincancer

TGFβ‐inducedEMThasbeendemonstratedtocontributetothemetastasisof

severalcancertypesincludingbreastandheadandneckcancer(Thieryetal.,2009;

Heldinetal.,2012).EMTincreasesmetastaticpotentialbyconvertingstationary,

transformedepithelialcellsintomotile,mesenchymalcancercells(Thieryetal.,

2009;Heldinetal.,2012).Overthepastdecademanystudieshaveexpandedthe

knowledgeofEMTandmetastasisinothertumortypesandalsocorroboratedthe

importanceofTGFβsignalinginthisprocess(reviewedbyHeldinetal.,2012).

IthasalsobecomewellestablishedthatEMTcanconferotherpro‐metastatic

propertiestocancercellsinparticularstemcelltraits,includingself‐renewal

capacity(Scheeletal.,2011).Thiswasfirstdemonstratedinimmortalizedhuman

mammaryepithelialcells(HMLE)whereforcedexpressionofEMTtranscription

factorsSnail1andTwist1resultsintheacquisitionofstemcelllikecharacteristics

(Manietal.,2008).Thisworkhasrapidlybeenexpandedandseveralimportant

discoverieshavebeenmadeincludingtheobservationthatthecytokinesTGFβ1and

Wnt5awhencombinedwithareductioninWntantagonists(Dickkopf‐1and

secretedfizzledrelatedprotein1)caninducedastemcellstatethroughtheEMT

withoutanygeneticmanipulationoftargetcells(Scheeletal.,2011).Furthermore,

EMTtranscriptionfactorSnail2wasdemonstratedtocooperatewithSox9to

positivelyregulatethemammarystemcellstateinnontransformedcells(Guoetal.,

2012).ThisrapidlyexpandingpoolofdatathatsuggestsEMTprogramcaninducea

28

stemcellstatehasimportantimplicationsformulti‐tissueregeneration.

EMTinregeneration

EMThasneverbeendefinitivelylinkedtomulti‐tissueregeneration,howeverthere

isagrowingbodyofevidencethatsuggestsitcouldbeanimportantplayerin

achievingregenerativecapacityaswellasdeterminingtheregenerationversus

fibrosisbalance.Inmodelsofmulti‐tissueregenerationEMTtranscriptionfactors

haveonlybeenobservedtwiceintheliterature(Satohetal.,2008;Kragletal.,

2012).Bothoftheseobservationsinvolvetheaxolotlmodeloflimbregeneration

whereitwasdemonstratedthatdifferentisoformsoftheEMTtranscriptionfactor

Twistareupregulatedduringregeneration(Satohetal.,2008;Kragletal.,2012).

TheauthorsofthesestudiesdonotmentionEMTintheirwork,butdoalludetothe

roleofTwistduringneuralcrestinductionanddelamination(Satohetal.,2008;

Kragletal.,2012).TheobservationofEMTtranscriptionfactorsduring

regenerationsuggestsayetunknownroleforthesefactorsintheprocess.

Speculating,withthecurrentresearchinmind,itislikelythattherolesEMT

transcriptionfactorsplayduringregenerationincludecellmotilityandcellfate

determination.Duringregeneration,cellsneedtomigratetoareasoftissuedamage,

thustheacquisitionofcellularmotilityislikelyaveryimportantdeterminantof

regenerativesuccess.However,inmodelsofblastema‐mediatedregeneration,a

morestrikingneedistheabilityforprogenitorpoolstorapidlyexpand.Ithasbeen

demonstratedinaxolotlsthatthisrapidexpansionoccursthroughde‐differentiation

29

ofexistingcellsinalineagerestrictedmanner,andthatpluripotentstemcellsare

notgenerated(Kragletal.,2009;Rinkevichetal.,2011).Notably,thisrapid

expansionofprogenitorsisalsoblockedwhenTGFβsignalingisinhibited(Levesque

etal.,2007;Jazwinskaetal.,2007;HoandWhitman,2008).Aforementionedwork

inthecancerresearchfieldhasdemonstratedthatthetransfectionofbothnormal

andtransformedmammarycellswithEMTtranscriptionfactorsSnail1,Snail2or

Twistcausesadedifferentiationandsubsequentacquisitionofstemcellliketraits

suggestingEMTtranscriptionfactorscanactaslineagerestrictedreprogramming

agents(Manietal.,2008;Guoetal.,2012).Furthermore,recentworkindrosophila

hasdemonstratedthattheSnailfamilytranscriptionfactorWorniuiscontinuously

requiredinneuroblaststomaintainself‐renewalbyinhibitingpremature

differentiation(Laietal.,2012).Mechanistically,ithasbeenshownduringmuscle

developmentthatSnailbindsparticularE‐boxesblockingdifferentiationpromoting

transcriptionfactorsfrombinding,thusineffectblockingdifferentiationand

promotingprogenitorphenotypes(Soleimanietal.,2012).Takentogether,itis

temptingtospeculatethatEMTtranscriptionfactors,ifinducedbythecorrect

extracellularstimuli,couldcauseasubsetofcellstodedifferentiateandbecomea

lineagerestricted,tissuespecificprogenitorsthatcoulddriveregeneration.

Theleopardgecko(Eublepharismacularius)modelofscarfreemulti­tissueappendageregeneration;

Mentionedpreviously,manylizardssuchasEublepharismaculariusandAnolis

30

carolinensiscanundergopostamputationregenerationoftheirtailsfollowingthe

voluntarysheddingoftheirtailsinaprocesstermautotomy(BellairsandBryant,

1985).Tailautotomyhasdevelopedevolutionarlyasananti‐predationstrategyand

hasbeenextensivelyexaminedintermsofecologicalcostsandbenefits(Nayaetal.,

2007).Morerecently,tailautotomyandsubsequentregenerationhasbeguntobe

examinedasamodelofscar‐free‐multi‐tissueappendageregeneration.Todate,

mostresearchexaminingmulti‐tissueregenerationinvertebrateshasfocusedon

anamniotessuchasXenopus,zebrafishandaxolotls.Modelsstudyingamniote

regenerationarehoweverbecomingmorecommonwithdigittipregenerationand

earwholepunchclosurebeingstudiedinmice,primates(suchashumans)and

rabbits(Bielefeldetal.,2012).OneofthemajorbenefitsoftheEublepharis

maculariusmodelofmultitissueregenerationisthatthecompleteregenerationofa

large,complexappendagesuchasthetail,hasnotbeenreportedinanyother

amniotes(McLeanandVickaryous,2011).Furthermoreithasbeenobservedthat

tailregenerationintheleopardgeckoinvolvestheformationofamassofnon‐

differentiated,highlyproliferativecellsresemblingtheblastemaseenfrequentlyin

anamniotesmodelsofregeneration(McLeanandVickaryous,2011).

Theregenerativeresponsefollowingtaillossintheleopardgeckohasbeen

documentedtofollowaconservedseriesofmorphologicalandhistologicalevents

thathavebeencharacterizedanddevelopedintoastagingsystem(McLeanand

Vickaryous,2011).Theoriginalgeckotailisacomplex,multi‐tissueappendagethat

containsnumeroustissuetypesincludingstriatedmuscle,adiposetissue,

31

vasculature,abonyvertebralcolumn,spinalcord,notochord,epidermis,anddermis

andcanrepresentupto41%oftotalbodylength(McLeanandVickaryous,2011).

Furthermore,thegeckotailhasuniqueadaptationsdesignedtofacilitateautotomy

includingintervertebralfractureplanesandproximalvascularsphincterstoreduce

bloodloss(McLeanandVickaryous,2011).

Postautotomymorphology:Woundhealing:stagesI­III

Immediatelyfollowingtailloss(StageI)theamputationplaneisanopenwound

withvarioustissuesexposedincludingvertebrae,spinalcord,dermis,muscle,and

adiposetissue.Duetothearterialsphinctersthereisrelativelylittlebloodloss.

From18hourstoupto8dayspostautotomy(StageII)anexudateclotcontaining

tissuedebris,erythrocytesandserumformsovertheexposedtissues.Notably,

duringthisstageepidermialcellsbeingtomigrateacrossthewound,deeptothe

exudateclot,toformwhatwillbecomethewoundepidermis.Theseepidermalcells

areproliferative(PCNApositive).Deeptothewoundepidermisanddistaltothe

spinalcord,anotherpopulationofcellsisalsodemonstratedtobeproliferative,

possiblyindicatingearlyblastemacells.StagesItoIIIaregroupedtogetherasthe

woundhealingphase(McLeanandVickaryous,2011).From4daysto8post

autotomythelossoftheexudateclotisobservedmarkingstageIIIofregeneration.

Duringthisstagenoregenerativeoutgrowthisobserved,however,thewound

epitheliumcoverstheentirewoundsurfaceandbeginstothickenandstratify

suggestingtheformationoftheapicalectodermalcap(AEC).Interestingly,

32

epidermalprotrusions(epidermaldowngrowths)alsobegintoappearlateraltothe

woundedges,whicharehighlyproliferative,migrateproximallyandframethe

blastema,whichbytheendofstageIIIisalsohighlyproliferativeandwell‐

established,deeptothewoundepitheliumanddistaltothespinalcord.

Postautotomymorphology:Regeneration:StagesIV­VII

FollowingstageIII,from8to15dayspostautotomythereisapronounced

outgrowthoftheregenerativetail(stageIV).Duringthisstage,theblastema

becomesalargeandpronouncedmassofproliferativecellsthatconstitutesmostof

theoutgrowthseen.Notably,thewoundepidermisbecomesmuchthickerthan

normal,adjacentepidermisandtheepidermaldowngrowthsbecomemore

prominent.Furthermore,stageIVmarkstheinitiationofregenerativemyogenesis

withcondensationsofmyoblastsappearingtobemoredifferentiatedinaproximal

(mostdifferentiated)todistal(leastdifferentiated)pattern.Fromday12to26post

autotomy(stageV)thetailcontinuestolengthenandregenerate.EarlyinstageV,

theblastemaremainsprominentbutdifferentiationappearstobeoccurringina

proximaltodistalfashionasacone‐likecondensationofputativecartilagecells

begintodepositECMcomponentsandcomponentsofthenervoustissuesuchas

axonsappear.Myogenesiscontinuestooccurinaproximaltodistalpattern,with

regeneratemyotubesbecomingmoredistinct.TowardstheendofstageV,the

woundepitheliumbeginstobecomekeratinizedandundergoesscalation.

Differentiationintheblastemabecomesmorepronouncedwithpresumptive

33

cartilagecellsdifferentiatingintoSox9expressingchondrocytesandchondroblasts

againinaproximaltodistalgradation(McLeanandVickaryous,2011).From18to

30dayspostautotomy(stageVI)theregeneratetailcontinuestodifferentiatewith

thewoundepidermisbecomingthesamethicknessasnormalepitheliumandmost

blastemacellsdifferentiatingintonumerous,definedtissues.Finally,fromday25

onward(stageVII)theregenerationprogrambecomescomplete,withthe

regeneratetailachievingataperedshapeandpigmentation,andmostblastemacells

differentiated.Thefullyregeneratetail,thoughsimilartotheoriginalstructure,is

notaperfectreplicaastheskeletalelementsoftheoriginaltailarereplacedbya

cartilaginousconeand,importantly,donotundergoskeletalmineralization

(McLeanandVickaryous,2011).Despitetheextensivemorphologicaland

histologicalcharacterization,tailregenerationinEublepharismaculariushasnot

beenextensivelyexaminedatthemolecularlevel.Preliminaryanalysisofthescar

freewoundhealingprocesshasdemonstratedthatTGFβ3ispresentonlylate(stage

Vonward)duringgeckotailregenerationandislocalizedtochondrocytesofthe

regeneratingcartilaginouscone(McLeanandVickaryous,2011;Delormeetal.,

2012).

34

Rationale

TGFβ/activinsignalinghasbeendemonstratedtoplayavarietyofimportant

rolesduringbothscarformationandscar‐freewoundhealingleadingto

regeneration.Availableevidencestronglyindicatesthatthereparativeoutcome

(scarificationversusregeneration)isdeterminedbythespatiotemporalexpression

ofvariousTGFβandactivinligands.Thegoalofthisresearchwastodeterminethe

stageofonsetandlocationofexpressionofvariousTGFβ/activinsignalingligands

andpotentialdownstreamtargetsduringtheprocessoftailreplacementusinga

regeneration‐competentamniotemodel,theleopardgecko(Eublepharis

macularius).

IhypothesizethatligandsoftheTGFβ/activinsignalingpathwayplaydistinctive

rolesindifferentcellpopulationsandatdifferentstagesofthescar‐freewound

healingandtailregenerationprogram.

Objective1

Toestablishthatthecanonical(SMAD‐mediated)TGFβ/activinpathwayisactivated

duringgeckotailregenerationanddetermineiftheblastemalcellpopulationis

activelyrespondingtoTGFβ/activinsignalingbydocumentingtheonsetof

inductionandcellularlocalizationofphosphorylatedSMAD2(pSMAD2),andits

potentialco‐localizationwiththeproliferation,andsubsequentblastemamarker

PCNA,usingwesternblotanalysesandimmunofluorescence.

35

Objective2

InvestigatetheexpressionandlocalizationofTGFβ/activinfamilymemberscapable

ofactivatingtheTGFβ/activinsignalingpathwayduringgeckotailregeneration.

Morespecifically,bydeterminingproteinandmRNAexpressionofTGFβ1,TGFβ2

TGFβ3andactivin­βAthroughtheuseofwesternblotting,immunohistochemisty

andqRT‐PCR.ThisincludesthedeterminationofgeckomRNAsequences

(unavailabletodate)foreachofTGFβ1,TGFβ2,TGFβ3andactivin­βAthroughPCR

basedapproaches.

Objective3

ToinvestigatethepotentialroleofnovelTGFβ/activininducedcellularprogramsin

particular,theepithelialtomesenchymaltransition(EMT)duringgeckotail

regeneration(stagesIII‐VI)bydeterminingtheexpressionpatternofEMT

transcriptionfactorsknowntobeinducedbyTGFβ/activinsignaling,specifically

Snail1,Snail2,andZeb2usingqRT‐PCR.

36

MaterialandMethods

Alistofrecipesforsolutionspreparedandsuppliersforchemicalsarefoundin

appendicesI,andII,respectively.PrimersusedforPCRreactionscanbefoundin

Tables1and2inappendixIII.

AnimalCareandHandlingAllanimalswerecaptivebredandobtainedfromacommercialsupplier(Global

ExoticPets,Kitchener,Ontario).Leopardgeckoswerehousedindividuallyin

standardratenclosuresintheCentralAnimalFacilityattheUniversityofGuelph,

followinghusbandryprotocolsofMcLeanandVickaryous(2011)andstandard

animalcareproceduresinaccordancewiththeCanadianCouncilonAnimalCare

(CCAC)Guidelines(Animalutilizationprotocolnumbers09R026and10R113,

approvedbytheUniversityofGuelphAnimalCareCommittee).

TailCollectionandTissueSegmentationTailcollectionwasachievedbyautotomyusingtheprotocolofMcLeanand

Vickaryous(2011).Briefly,leopardgeckosweremanuallyrestrainedandtailswere

firmlypinchedatapositionhalfwaybetweenthecloacaandtailtip.Following

pinching,thedistalportionofthetailisself‐detached,whereastheproximalportion

isretained.Followingautotomy,tailswereallowedtoregeneratetoappropriate

stagesandautotimizedasecondtime.Secondarilydetachedtailsincludethe

regeneratedtissuesplusaportionoforiginaltail.Followingthesecondautotomy,

37

tailweresegmented(bytransversecutsusinga#15bladescalpelblade)intothree

domains:regeneratedtissue;asegment(~1.0cmlong)oforiginaltissue

immediatelyadjacenttotheregeneratedtissue(‘distaltissue’);andthenext

consecutivesegmentoforiginaltissuemoreproximaltothebody(‘proximal

tissue’).Eachsegmentwasimmediatelyflashfrozeninliquidnitrogenandstoredat

‐80forproteinandRNAextraction.

WesternBlotanalysisProteinwasextractedfromeachsegmentofindividualautotomizedtailsand

homogenizedinproteinlysisbuffer(CellSignaling),supplementedwith1mMPMSF,

2µg/mlaprotinin(Sigma‐Aldrich),1mMNa3VO4(NewEnglandBiolabs)and1%

phosphataseinhibitorcocktail2(Sigma‐Aldrich).Foreachsegment,a30µgsample

oftotalproteinlysatewasresolvedon10%and15%polyacrylamidegelsunder

reducing(pSMAD2,SMAD2,α‐Tubulin)ornon‐reducingconditions(TGFβ1)and

subsequentlytransferredtoPVDFmembranes(Roche).Membraneswerefirst

blockedfor1hourinTBSTcontaining5%skimmilkpowderandthenincubated

withprimaryantibodydilutedinblockingsolutionovernightat4°C.The

concentrationsofprimaryantibodieswereasfollow:1:1,000rabbitmonoclonal

anti‐pSMAD2(Ser465/Ser467)(CellSignaling),1:1,000mousemonoclonalanti‐

SMAD2(CellSignaling),1:500,000mousemonoclonalanti‐tubulin(Sigma‐Aldrich),

1:1,000rabbitpolyclonalanti‐TGFβ1(SantaCruz).Membraneswerethenwashed

withTBST,incubatedwiththeappropriateHRP‐conjugatedsecondaryantibodies

for1houratroomtemperature,washedagainwithTBSTanddevelopedwithHRP

38

chemiluminescentsubstrates(Millipore)visualizedonaChemiDocXRS(Bio‐Rad).

Threebiologicalreplicateswereexaminedbywesternblottingforeachproteinof

interest.

ImmunofluorescenceandImmunohistochemistryFixationandsectioningwereperformedaspreviouslydescribed(Mcleanand

Vickaryous,2011).Briefly,tissueswerefixedineither10%neutralbuffered

formalin(NBF)or4%paraformaldehyde(PFA)andseriallysectioned.Slide

mountedserialsectionswerethendeparaffinizedandrehydrated.Sectionsdestined

forimmunohistochemicalanalysiswerequenchedin3%H2O2for30minutes.All

sectionswererinsedwithphosphatebufferedsaline(PBS).Next,heatinduced

epitoperetrieval(citratebufferheatedto90°Cfor12minutes)wasusedtounmask

antigenicsites.Followingantigenretrieval,sectionswereincubatedwith

appropriateblockingreagentforonehour,followedbyprimaryantibodyovernight

at4°C.Primaryantibodyconcentrationswere1:150anti‐pSmad2(Ser465/467)

(CellSignalling),1:300anti‐PCNA(Dako),and1:500anti‐TGFβ1(SantaCruz).

Primaryantibodywasomittedfromonesectiononeachslidetofunctionasa

negativecontrol.Forimmunohistochemistry,slideswererinsedinPBSandthenall

sectionswereincubatedwithappropriatebiotinylatedsecondaryantibody(Vector

Laboratories)ataconcentrationof1:500.Slideswerethenincubatedwith

peroxidase‐streptavidinsubstrate(VectorLaboratories)for1hratroom

temperature,andvisualizedwith3‐3’‐diaminobenzidine(DABfromVector

Laboratories;dilutedin5mldH2O,for6minutesperslide).Slideswerethenrinsed

indH20,counterstainedinMayersHematoxylinfor4minutesandmountedwith

39

coverslips.Forimmunofluorescence,secondaryantibodyconcentrationsand

incubationswere1:300Alexa‐Fluorgoatanti–rabbit488(LifeTechnologies),or

1:300Cy3Goatanti‐mouse(JacksonImmunoResearchLaboratories)foronehourat

roomtemperature.Followingtheadditionoffluorescentsecondaryantibodies,

slideswereincubatedwith0.3µM4’,6’–diamidino‐2‐phenylindole(DAPI)(Sigma)

dilutedinPBSfor4minutes,washedtwiceandmountedwithaqueousmounting

mediaandcoverslips.

IdentificationofEublepharismaculariuscDNAsequencesusingdegenerativePCRRNAwasisolatedfromsegmentatedtailsfrozenat‐80byhomogenizationinTrizol

(LifeTechnologies)atcoldtemperaturefollowedbytheAurumtotalisolationRNA

kit(Bio‐Rad).cDNAsynthesiswasperformedusingtheiScriptcDNAsynthesiskit

(Bio‐Rad)with1mgtotalRNA.Degenerateprimersweredesignedbyeyebasedon

conservedsequencesidentifiedusingClustalW(EMBL‐EBI)andpubliclyavailable

sequencesfromrelatedspecies.(ForprimersequencesseeTables1and2).PCRwas

performedonaCFX‐96thermocycler(Bio‐Rad)usingSYBRGreenISupermix

(AppliedBiosystems)acrossvarioustemperatures.Ampliconswererunona2%

agarosegelandcorrectlysizedbandsweresubsequentlyextractedusingillustra

GFXPCRGelBandPurificationKit(GEHealthcare).Followingextraction,amplicons

wereeithersentforsequencingattheadvancedanalysisfacilityoftheUniversityof

GuelphorclonedintoaTOPO‐TAcloningvectorpGEM‐TEasy(Promega)andthen

sequenced.SequenceinformationwasthenanalyzedusingNCBInucleotideblast

andClustalW(EMBL‐EBI)toconfirmsequencespecificity(Table3).Identified

40

sequencesweredepositedtoNCBIGenBank(numberstobeaddedoncesequences

annotated).

qRT­PCRanalysisRNAwasisolatedandcDNAwasproducedasdescribedabove.Appropriatequality

controlstepswereincludedalongthewayconformingtotheMIQEguidelines

(Bustinetal.,2009;Tayloretal.,2010).ThecDNAthatwasobtainedwasdiluted

1:4innucleasefreewaterand4µlofdilutedcDNAwasaddedtoreactionmixes(10

µl)containing5µlSsoFastEvaGreenSupermix(Bio‐Rad)and500nMofeach

primer.PrimersweredesignedusingPrimer3(NCBI)andoptimizedusinga

temperaturegradientandeightpointstandardcurvetodeterminePCRefficiency.

Acceptableefficiencywasdeemedbetween90%and110%.Allampliconswere

determinedtobespecificbyagarosegelanalysisandsubsequentsequencing.qRT‐

PCRamplificationswerecarriedoutusingaCFX‐96(Bio‐Rad)asfollows:aninitial

denaturationstep2minutesat95°C,followedby40cyclesof5secondsat95°Cand

5secondsat59°C.Datawasexpressedasrelativegeneexpressionnormalizedto

18smRNAwhichwasdeterminedtobeasuitablehousekeepinggeneusingqBase

Plussoftware(BioGazelle).Kruskal‐Wallisnon‐parametricstatisticswere

performedonqRT‐PCRdatafollowedbyaDunnsposttestwithap‐valuecutoffof

>0.05.Threebiologicalreplicatesandthreetechnicalreplicateswereusedforall

qRT‐PCRexperiments.

41

Results

TGFβ/activinsignalingisactiveintheregenerateblastema.

Theblastemaischaracterizedbyahighlyproliferativepopulationofcells

contributingtotheformationofreplacementtissue(Stoick‐Cooperetal.,2007;

StocumandCameron,2011).Todetermineifcellscompromisingthepresumptive

blastemaareactivelyrespondingtotheTGFβ/activinsignalsweperformeddual

immunofluorescencewithpSMAD2andtheproliferationmarkerproliferatingcell

nuclearantigen(PCNA)atseveralstagesoftailregeneration.

Duringthelatewoundhealingphaseoftailregeneration(stageIII),presumptive

blastemacellsformasanaggregationofmesenchymallikecellsatthedistalendof

thetornspinalcord(Fig.1.A).InstageIIIregeneratetailsPCNAlabelsthissame

populationofmesenchymallikecellsdistaltothetornedgeofthespinalcord

representingtheinitiationoftheblastema(Fig.1.B).IsolatedPCNApositivecells

arealsodetectedintheregeneratedermislateralanddistaltothespinalcord(Fig.

2.).pSMAD2expressionappearsinnumerousnucleiofcellswithinthespinalcord

aswellasalargeportionofcellsintheinitialblastemalaggregation(Fig.1.C).PCNA

andpSMAD2co‐localizetonumerouscellswithinthePCNApositiveblastema

locateddistaltothespinalcordsuggestingactiveTGFβ/activinsignalingwithin

earlyblastemacellpopulations(Fig.1.D)

Followingthewoundhealingphase(stageIII)theregeneratingtailbeginstogrow

42

distallyandformacone‐likeoutgrowthoftenreferredtoastheregeneratetailbud

(stageIV).Thisoutgrowthismostlyattributedtoalargeincreaseinthenumberof

mesenchymallikeblastemacellslocateddistaltothespinalcord(stageIV)(Fig.1.

E).PCNAlabelsalargeportionoftheseblastemacellssupportingthedefinitionof

thegeckoregenerateblastemaasaproliferatepoolofmesenchymallikecells(Fig.1.

F).Interestingly,theregenerateepitheliumappearstolackabasallyrestricted

proliferativepopulationandhasbecomestrikinglyhyperthickenedasitresemblesa

structureknownasthewoundepithelium(Fig.1.F).pSMAD2nuclearlocalization

appearsinmostcellslocatedintheregeneratetailbudsuggestingextensive

TGFβ/activinsignalswithinearlyregeneratetailtissues(Fig.1.G).MostPCNA

positivecellswithintheregeneratetailbuddoublelabelforpSMAD2suggesting

thatblastemacellsareactivelyrespondingtoTGFβ/activinsignalsinearly

regeneratetissues(Fig.1.H)

Asregeneratetailoutgrowthcontinues(stageV)theblastemalcellpopulation

furtherincreasesinsizetosupporttailgrowth(Fig.1.I).PCNAlabelsmostcells

throughouttheregeneratetailbuddemonstratingalargeblastemaproximaltothe

woundepithelium(Fig.1.J).pSMAD2stainingagainappearsrobustthroughoutthe

tailbudfurthersuggestingstrongTGFβ/activinsignals(Fig.1.K).Closerinspection

oftheregeneratetailbudrevealsnumerousdualpositivecellsindicatingagainthat

blastemacellsareactivelyrespondingtoTGFβ/activinsignals(Fig.1.L)

Astheregenerativeoutgrowthbeginstoslowdown(stageVI)tissuedifferentiation

43

beginstobecomemoreapparent.Particulartissues,suchasregeneratingmuscle

andcartilage,becomehighlyvisibleindistalregeneratetissuesatstageVI(Fig.1.

M).PCNAexpressionbecomesmuchmorelocalizedtotissuespecificcompartments

instageIVtailssuchaslateralcartilaginouscellsofthecartilagecone(Fig.1.M).

FewerPCNApositivecellsarealsoobservedinthedermalcompartmentfurther

supportingblastemalcelldifferentiation(Fig.1.N).Furthermore,thedistalwould

epitheliumisnolongerhyperthickened,suggestingmaturationofwoundepithelium

tonormalepithelium.pSMAD2stainingremainsrobustthroughouttheregenerating

tailbudlikelyowingtotheimportantroleofTGFβ/activinsignalingincartilage

developmentandtissuedifferentiation(Fig.1.O).PCNAandpSMAD2appeartoco‐

localizetofewercellswithinthemesencymalcompartmentoftheregeneratetail,

supportingthenotionofblastemaldifferentiationatstageVI(Fig.1.P).These

observationsdemonstratethatTGFβ/activinsignalsarehighlyactivethroughout

theregeneratetailbudinearlystagesoftissueregenerationandthatblastemacells

areactivelyrespondingtotheseTGFβ/activinbasedsignals.

Phosphorylated­SMAD2andPCNAexpressioninoriginalandregenerateepidermis

Duringmostinstancesofmulti‐tissueregenerationauniquestructureknownasthe

woundepitheliumformsdistaltotheblastema.Intheleopardgeckothiswound

epitheliumbecomesapparentatstageIIandstageIIIwhereepithelialcellsmigrate

tocovertheautotomywoundandsubsequentlyundergoproliferation.StageIII

woundepitheliumisnormalthicknessbutdemonstratesalackofabasally

44

restrictedPCNApopulationofkeratinocytes(Fig.2.A)pSMAD2expressionis

detectedthroughoutthewoundepitheliumatstageIII(Fig.2.B).MovingtostageIV

thewoundepitheliumbecomeshyperthickenedwithPCNAlabelingapopulationof

suprabasalkerationcytes(Fig2.C).pSMAD2expressionisdetectedthroughoutthe

epitheliumbutappearstobeabsentinthemostbasallayers(Fig.2.D).StagesV

revealsahypertickenedepitheliumsimilartostageIVwithPCNAandpSMAD2

beingexpressedbysuprabasalkeratinocytes(Fig.2.E,F).Asregenerationproceeds

(stageVI)tissuedifferentationbeingstooccurandthewoundepitheliumbecomes

normalthickeness.PCNAbeginstobeexpressedinbasalkeratinocytesofthestage

VIepitheliumandpSMAD2isobservedsuprabasaly(Fig.2.G,H).Original

epitheliumintheleopardgeckodemonstratesabasallyrestrictedPCNApopulation

(Fig2.I)aswellaspSMAD2expressiondetectedthroughouttheepidermis(Fig.2.

J).Interestingly,examiningthePCNAexpressioninthelateralaspectsofastageV

tailsrevealsthepointatwhichthenormalepidermisandwoundepidermis

transition(Fig.2.L).Movinglefttoright(Fig.2.L),PCNAexpressionisinitially

expressedbybasalkeratinocytesbutrapidlybecomessuprabasalmovingtowards

theregeneratetissue.TheseresultsrevealthatexpressionofPCNAandpSMAD2in

thewoundepitheliumoftheleopardgeckoisdynamicanddifferentthanthatofthe

originalepithelium.

Phosphorylated­SMAD2isupregulatedinregeneratingtails.

ToconfirmthatthecanonicalTGFβ/activinpathwayisactivatedduringtail

regeneration,weinvestigatedtheproteinexpressionofpSMAD2,nativeSMAD2as

45

wellastheTGFβligandTGFβ1usingwesternblottingofsegmentedregeneratetails

(α‐tubulinusedasaloadingcontrol)(Fig.3.A‐D).InstageIII,IV,VandVItails

pSMAD2isdetectedmoststronglyinthedistalregeneratetissueconfirmingrobust

activationofthecanonicalTGFβ/activinsignalingpathwayinthetissuesofthe

regeneratetailbudandblastema(Fig.3.A‐D).pSMAD2isnotdetectedinoriginal

tailtissueandisonlyweaklyobservedinmoreproximaltissuessections(Fig.3.A‐

D).NativeSMAD2isexpressedrelativelyevenlyacrossalltissuesexamined,asis

ourloadingcontrolα‐tubulin(Fig.3.A‐D).TGFβ1appearstobeexpressedrelatively

evenlyacrossalltissueswiththenotableexceptionofthemostdistalregenerate

tissueinstageIVtails(Fig.3.B).TheseobservationsconfirmthatpSMAD2is

stronglyinducedinalldistalregeneratetissuesfurtherdemonstratingrobust

TGFβ/activinsignalingintheregeneratetailbud.ThisdataalsosuggeststhatTGFβ1

maynotbethepSMAD2inductiveligand,inparticularatearlystagesof

regeneration(stageIV).

TGFβ1expressionduringtailregeneration.

TofurtherconfirmthisconspicuousabsenceofTGFβ1atstageIVweperformed

immunohistochemistryforTGFβ1atnumerousstagesofregeneration(stageIII,IV,

V,VI)andoriginaltailtissue(Fig.4).Inoriginaltissuefrombothproximaltail

sectionsaswellasnon‐regeneratetailsTGFβ1expressionisobservedstrongly

throughouttheepidermis(Fig.4.A).TGFβ1expressionisalsoobserved

surroundingnumerousmesenchymal‐likedermalcells(Fig.4.A:cellsidentified

46

withMandarrows).InstageIIIregeneratetissueTGFβ1expressionisobserved

weaklyintheregeneratewoundepidermisaswellasinavarietyofputative

immunecellslocatedinthedermisproximaltotheautotomyplane(Fig.4.B:cells

identifiedwithIandarrows).Strikingly,instageIVregeneratetissueTGFβ1

expressionisnearlyabsentfromthewoundepidermisaswellastheproximal

blastemalpopulationconfirmingourwesternblotdatainFigure3(Fig.4.C).As

regenerationproceeds(stageV)TGFβ1expressionbeginstobeobservedinlateral

aspectsofthewoundepitheliumaswellassurroundingnumerouscellswithinthe

blastema(Fig.4.D).BystageVITGFβ1expressionhasreturnedtothematuring

woundepithelium(Fig.4.E).Furthermore,theexpressionofTGFβ1bycellswithin

thestageVIdermisbeingstoresemblethatoforiginaltissue(Fig.4.E).The

immunohistochemicallocalizationofTGFβ1duringtailregenerationfurther

confirmstheobservationthatTGFβ1islargelyabsentfromearly(stageIV)

regeneratetissue,andasregenerationproceedsTGFβ1expressiongradually

returnstoasregeneratetissuesdifferentiate.

Non­overlappingTGFβ1andpSMAD2expressioninearlytailregeneration.

TheobservationthatTGFβ1isabsentinearlyregeneratetissuedespitethelarge

amountofpSMAD2beingpresentsuggeststhepresenceofotherTGFβ/activin

ligands.Toconfirmthenon‐overlappingexpressionofTGFβ1andpSMAD2we

examinedtheexpressionofTGFβ1andpSMAD2onadjacenttissuesections(Fig.5).

InstageIVregeneratetailsastructureknownastheepidermaldowngrowthforms

47

andappearstoseparateoriginaltissuefromtheregenerateblastema(Mcleanand

Vickaryous,2011)(Fig.5).OnthelateralsideoftheepidermaldowngrowthTGFβ1

expressionisobservedintheepitheliumaswellasnumerousdermalcells(Fig.5.A,

B).However,onthemedialsideoftheepidermaldowngrowthTGFβ1expressionis

undetectableinregeneratetissues(Fig.5.A,C).pSMAD2expressionisdetectedin

numerousTGFβ1expressingcellsoftheepidermisanddermiswithintheoriginal

tissuelateraltotheepidermaldowngrowth(Fig.5.D,E).Interestingly,inregenerate

tissuesmedialtotheepidermaldowngrowthwhereTGFβ1expressionisnot

observedthereappearstobealargeincreaseinthenumberofpSMAD2positive

cells(Fig.5.D,F).ThisresultconfirmsthereciprocalexpressionofTGFβ1and

pSMAD2inoriginalandregeneratetissuesandsuggestsanotherTGFβ/activin

ligandispresentinearlyregeneratetissues.

Activin­βAisupregulatedintheblastemaduringearlystagesoftailregeneration.

ToidentifytheunknownpSMAD2inducingligand(s)atstageIVofregenerationwe

performedaqRT‐PCRscreenforpSMAD2activatingligandsinregeneratetissue

fromstageIVblastemas.Toperfomthisscreenwefirstclonedallthreemembersof

theTGFβ/activinfamily,TGFβ1,TGFβ2,TGFβ3aswellasactivin­βAwhichare

knowntoclassicallyactivatepSMAD2aswellastheTGFβsuperfamilymemberbone

morphogenicprotein2(BMP2)(MoustakasandHeldin,2009).Wethenexamined

mRNAexpressionofthesepSMAD2activatingligandsusingqRT‐PCRonsegmented

stageIVtails.Comparingproximal,distalandregeneratingtissueatstageIV,we

48

foundthatmRNAexpressionofTGFβ1andremainsrelativelyconstant(Fig.6)

demonstratingnotranscriptionalupregulationistakingplaceinthenewlyforming

tissuesandfurtherconfirmingourwesternblotandimmunohistochemistrydataon

TGFβ1.AlthoughTGFβ2,TGFβ3andBMP2revealslightupregulationinregenerating

tissueswithTGFβ3beingthelargest,thesefoldincreasesarenotstatistically

significant(Fig.6).Wedidhoweverobserveasignificant15‐foldupregulationin

activin­βAmRNAexpressioninregeneratingtissues(Fig.6).Theseresultspoint

towardsactivin‐Aasacandidateligandresponsibleforthelargeinductionof

pSMAD2duringtheearlystagesoftailregeneration.

IdentificationofnovelTGFβ/activintargetgenesupregulatedinregeneratetissues.

TGFβ/activinligandsparticipateinawiderangeofcellularprocesses,manyof

whicharelikelytoplayimportantrolesduringmulti‐tissueregeneration.We

investigatedtheTGFβ/activininducedtargetgenesSnail1,Snail2(formerlySlug)

andZeb2atstageIVtailsusingqRT‐PCR.Snail1,Snail2andZeb2aretranscription

factorsknowntomediatetheepithelialtomesenchymaltransition(EMT)aprocess

implicatedindrivingcellmotilityaswellasstemness(LimandThiery,2012).The

EMTprocesshasbeenimplicatedindevelopment,cancerandwoundhealingbut

hasneverbeensuggestedtoplayaroleinmulti‐tissueregeneration.Interestingly

weobservedasignificantupregulationofSnail1andSnail2(butnotZeb2)inthe

regeneratetissuesofthegeckotailcomparedtoregeneratetissues.(Fig.7).This

novelfindingcombinedwithourrobustpSMAD2expressionseenintheblastema

49

suggeststhattheepithelialtomesencymaltransitioncouldbeanimportant,

TGFβ/activinmediatedprocessthatisactiveduringmulti‐tissueregeneration.

50

Figure1.ImmunofluorescenceofPCNA(red)andpSMAD2(green)expressionduringfourstagesofgeckotailregenerationandassociatedschematicrepresentationsoflongitudinalsections.StagesIII(A­D)dottedlinesurroundsputativeblastemacellpopulationdistaltospinalcord.StageIV(E­H).StageV(I­L).StageVI(M­P)dottedlinesurroundscartilaginouscone.Allimagestakenat20xmagnification,scalebars=100µm.sc=spinalcord.cc=cartilaginouscone.

51

Figure2.ImmunofluorescenceofPCNA(red)andpSMAD2(green)expressionindistalwoundepitheliumandblastemaduringfourstagesoftailregeneration,originaltissue,andstageVtransitionalepithelium.StageIII(A­B).StageIV(C­D).StageV(E­F).StageVI(G­H).Originaltissue(I­J).StageVtransitionalepithelium(L­M),arrowmarkstransitionbetweenoriginalepithelium(leftside)andwoundepithelium(rightside).Allimagestakenat40xmagnification,scalebars=200µm.bv=bloodvessel,cc=cartilaginouscone.

52

Figure3.Representativewesternblotsanalysisofsegmentedgeckotailsatfourstagesoftailregenerationaswellastissuefromoriginaltails(n=3).StageIII(A).StageIV(B).StageV(C).StageVI(D).NotetheinductionofpSMAD2inmostdistalregeneratetissueatallstagesofregenerationandtheconspicuousabsenceofTGFb1proteinexpressioninstageIVregeneratetailtissue.NativeSMAD2anda‐tubulinremainrelativelyconstantandserveasloadingcontrols.

53

Figure4.ImmunohistochemicallocalizationofTGFβ1proteinexpressionduringfourstagesofgeckotailregenerationaswellasoriginaltissue,positivesignalisdemonstratedbybrownstaining(DABchromogen).Negativecontroltissueshownontherighthandpanel.Originaltissue(A,An).StageIII(B,Bn).StageIV(C,Cn).StageV(D,Dn).StageVI(E,En).Scalebar=100µm.ArrowsmarkedM=TGFβ1positivemesechymallikecells.ArrowsmarkedI=TGFβ1positiveimmunecells.OE=Originalepithelium.WE=Woundepithelium.

54

Figure5.Non‐overlappingexpressionofTGFβ1andpSMAD2inoriginalandregeneratetissueinstageIVregeneratetails.TGFβ1expression(A)aswellashighmagnificationinsets(B,C)andnegativecontrols(Bn,Cn).pSMAD2expressiononadjacenttissuesection(D)aswellashighmagnificationinsets(E,F).Scalebarfor(A,D)=200µm,scalebarfor(B,C,E,F)=50µm.

55

Figure6.RelativemRNAlevelsofTGFβfamilymembersinsegementedstageIVregeneratetailsdeterminedusingquantitativereal‐timepolymerasechainreaction(qRT‐PCR)andnormalizedto18smRNAlevels.Graphdepictstheaveragefoldchange(±SEM)insegmentedtissuesectionsrelativetomosttheproximaltissuesection.Astatisticallysignificantincreaseinactivin­βAwasobservedinthemostdistaltissuesectioncorrespondingtonewlyregeneratetissueandthepositionoftheblastema.StatisticalsignificancewasdeterminedusingKruskal‐Wallisnon‐parametricstatisticsfollowedbyaDunnsposttestwithap‐valuecutoffofp>0.05.n=3forbiologicalandtechnicalreplicates.

56

Figure7.RelativemRNAlevelsofEMTtranscriptionfactorsinsegementedstageIVregeneratetailsdeterminedusingquantitativereal‐timepolymerasechainreaction(qRT‐PCR)andnormalizedto18smRNAlevels.Graphdepictstheaveragefoldchange(±SEM)insegmentedtissuesectionsrelativetomosttheproximaltissuesection.AstatisticallysignificantincreaseinSnail1andSnail2wasobservedinthemostdistaltissuesectioncorrespondingtonewlyregeneratetissueandthepositionoftheblastema.StatisticalsignificancewasdeterminedusingKruskal‐Wallisnon‐parametricstatisticsfollowedbyaDunnsposttestwithap‐valuecutoffofp>0.05.n=3forbiologicalandtechnicalreplicates.

57

Discussion

TheTGFβ/activinsignalingpathwayisactivatedthroughoutthetailregenerationprogram.

Amongamniotes,oneofthemostdramaticexamplesofspontaneousmulti‐tissue

regenerationistherestorationofthelizardtail.Followingtailloss(typicallyin

responsetothethreatofpredation),manylizardspeciesareabletorecreatea

replacementappendage,completewithskeletalsupport,nerves,muscleandaspinal

cord(BellairsandBryant,1985).Previousworkinanamniotes,namely,zebrafish,

axolotlandXenopushasinvestigatedtheTGFβ/activinsignalingeventsthatoccur

duringearlystagesoftailfinregeneration(Jazawinskaetal.,2007;Levesqueetal.,

2007;HoandWhitman,2008).WedeterminedthattheTGFβ/activinsignaling

pathwayisalsobeingactivatedduringtailregenerationingeckos.TGFβ/activin

signalingoccursthroughoutblastemaformation,regenerativeoutgrowthandtissue

differentiation,representingstagesIII‐VIoftheregenerationprogram.

PhosphorylationandsubsequentnuclearimportofSMAD2occursfollowingeither

TGFβoractivinligandsbindingtotheircognatereceptors(MoustakasandHeldin,

2009).ThenuclearlocalizationofpSMAD2withimmunofluroscenceservesas

robustreadoutofactiveTGFβ/activinsignallingwithinacell.Our

immunofluorescencedata(furtherconfirmedbywesternblot)demonstratesthat

pSMAD2expressionisrobustthroughouttheregeneratingtissuesoftheofthe

regeneratetailbud.Asevidencedbyimmunofluorescentco‐localizationofPCNA

58

andpSMAD2,wedemonstratethatalargeportionofputativeblastemacellsare

activelyrespondingtoTGFβ/activinsignalingthroughoutregeneration.

Interestingly,TGFβ/activinligandsareclassicallythoughtofas

antiproliferative/cytostaticcytokinesasSMAD2/3complexesareknowntoinhibit

cellcycleprogression(Sandhuetal.,1997;Seoaneetal.,2004;Alexandrowand

Moses,1995).However,duringdevelopmentaswellascancerprogressionthe

cytostaticfunctionsofTGFβsignalingcanbeovercomebyanumberofdifferent

mechanisms(Massague,2012).Cellularcontext,inparticular,crosstalkwithother

signalingpathwayshasbeendemonstratedtobeabletoovercometheTGFβ

mediatedcytostaticprogramthroughintegrationattheleveloftheSMADs(Guoand

Wang,2009;Massague,2012).Forexample,SMAD‐β‐catenincomplexesdrivenby

TGFβandWntsignallingpromotestemcellproliferationandself‐renewalby

stimulatingmitosis,whileinhibitingdifferentiation(Jianetal.,2006).Thus,the

abilityforSMADproteinstocooperatewithothersignalsanddirectdiverse

transcriptionaleffectsdependingonthecellularcontextrepresentsalogical

explanationfortheirconsistentlyobservedinvolvementinmulti‐tissue

regeneration.

Activin,andnotTGFβisupregulated.pSMAD2expressionisareliablereadoutofcanonicalTGFβ/activinsignaling

(MoustakasandHeldin,2009),butdoesnotdistinguishbetweenactivating

ligand(s).GiventhedocumentedrolesofTGFβ1duringmammalianwoundhealing

59

(FergusonandO’Kane,1994;Bielefeldetal.,2012),weinvestigatedthespatio‐

temporalexpressionofTGFβ1duringgeckotailregeneration.Ourwesternblotdata

demonstratesthatTGFβ1isconstitutivelypresentinoriginaltailtissueandatmost

stagesofregeneration(stagesIII,V‐VI).However,detectableTGFβ1was

conspicuouslyabsentfromregeneratingtissuesatstageIV.Wefurtherconfirmed

thisresultusingimmunohistochemistry,whereweobservedthatTGFβ1protein

expressionisstrikinglyabsentfromtheearlyregeneratetissues,includingthe

blastema,andwoundepidermis.Thisunusualresultsuggeststhattheabsenceof

TGFβ1fromearlyregeneratetissuesmaybearequirementforsuccessful

regeneration.TGFβ1isclassicallycharacterizedasapro‐fibroticcytokine.In

agreementwiththis,scar‐freewoundhealinginfetalmammalsischaracterizedby

lowerlevelsofTGFβ1andhigherlevelsofTGFβ3,whencomparedtoscarforming

wounds,whileinadultmammalsscarformationisdriven,atleastinpart,byan

increaseinTGFβ1(Sooetal.,2003;Larsonetal.,2010;Bielefeldetal.,2012).

Furthermore,whenscar‐freefetalwoundsaretreatedwithTGFβ1,scarification

occurs(LinandAdzick,1996).Thus,thelimitedexpressionofthisproteinis

consistentwithtailregenerationasascar‐freephenomenonandsuggeststhatthe

absenceofTGFβ1inearlystagesofregenerationmaybearequirementforscar‐free

woundhealing.

DespitetheabsenceofTGFβ1expressioninregeneratingtissuewestillobserveda

largeproportionofcellsoftheblastemaandwoundepitheliumactivelyresponding

toTGFβ/activinsignalsasevidencedbynuclearpSMAD2.Inordertoinvestigatethe

60

ligandsresponsibleforthisSMAD2phosphorylationinregeneratetissueweused

qRT‐PCRtoscreenforTGFβ/activinmRNAtranscriptsupregulatedinregenerate

tissues.WedeterminedusingourqRT‐PCRscreenthatthereisasignificantincrease

inactivin­βAmRNAbutnosignificantupregulationofeitherTGFβ1,TGFβ2,TGFβ3

orBMP2mRNAinearlyregeneratetissues.Thesefindingsindicatethatactivin‐A,

andnotTGFβ1orotherTGFβligands,islikelytheprimarydriverofSMAD2

phosphorylationduringearlystagesoftailregeneration.

Previousstudiesonzebrafishfinregenerationhavealsoreportedthatactivin­βA

mRNAisalsotheonlyTGFβ/activinligandsignificantlyupregulatedinregenerate

fintissues(Jazwinskaetal.,2007).Thisincreaseinactivin­βAexpression,combined

withaconspicuousabsenceofTGFβ1,suggeststhatthetypeandlevelsofthe

TGFβ/activinisoformsexpresseddeterminesregenerationoutcomeandpoints

towardsactivin‐mediatedsignalingasanimportantmediatorofbothscar‐free

woundhealingandtheestablishment(ormaintenance)ofcellproliferationduring

multi‐tissueregeneration.Insupportofthis,studiesinchickgranulosacells

demonstratedthatactivin‐AtreatmentcooperateswithproteinkinaseA(PKA)

activitytocausearapid,exclusiveinductionofSMAD2mRNAandprotein

(Schmiereretal.,2003).ThisresultsinashiftintheSMAD2/SMAD3ratiowithina

respondingcellthatpreferentiallyincreasesSMAD2phosphorylationandSMAD2

mediatedtranscription(Schmiereretal.,2003).Complementarytothis,several

studieshavedemonstratedthatSMAD2andSMAD3targetasubsetofseparate

genesresultingindifferenttranscriptionalandcellulareffects(Kretschmeretal.,

61

2003;Dzwoneketal.,2009;Petersonetal.,2009).Further,SMAD2knockoutmice

areembryoniclethalwhereasSMAD3miceareviableandfollowingwounding,

SMAD3knockoutmiceshowreducedECMdeposition,fewerfibroblastsanda

smallerwoundareawhencomparedtocontrolmice(Ashcroftetal.,1999).Taken

together,thesefindingssuggestthatbyselectivelyactivatingSMAD2,potentially

throughactivinsignaling,fibroticresponsescanbeattenuated.Further

investigationofTGFβ/activinsignalingduringtissueregeneration,particularlyon

thefunctionofactivin’sinmodulatingtheSMAD2/3balanceandsubsequent

transcriptional/cellularoutcome,isnecessarytoestablishactivin’sroleinscar‐free

woundhealing.

EMTtranscriptionfactorsareupregulatedduringtailregeneration.

ThelargepSMAD2expressionseenthroughoutregeneratetissuescombinedwith

previousworkshowingthatwhenSMAD2phosphorylationisblockedregeneration

cannotproceedsuggeststhatSMAD2mediatedtranscriptionalprogramsunderly

regenerateability.TGFβ/activinmediatedSMAD2signalingcaninduceavarietyof

transcriptionaleffectsdependantoncellularcontext,suchassignallingpathway

crosstalk(GuoandWang,2009).BasedontheobservationsbyKraglandothersthat

therapidexpansionoftissuespecificstemcells,likelythroughdedifferentiationof

adultcells,underliesregenerateabilitywedecidedtoexamineTGFβ/activin

inducedepithelialtomesenchymaltransition(EMT)transcriptionfactorsduringtail

regeneration.EMTtranscriptionfactorsacttoconvertstationaryepithelialcellsinto

62

amotilemesenchymal‐likephenotype.Duringdevelopment,EMTservesasan

essentialmechanismguidingseveralphasesofmorphogenesisandorganogenesis

includinggastrulationandneuralcrestdelamination(Hay,1995;LimandTheiry,

2012).Inadults,stimulationoftheEMTprogramisknowntopromoteorgan

fibrosis(KalluriandNeilson,2003)andtumormetastasis(Thiery,2002).Recently

however,ithasbeenshownthatEMTtranscriptionfactorscanalsoinduceastem

cell‐likephenotypeinbothnormalandtransformedcelllinesinvitro(Manietal.,

2008;Guoetal.,2012)andblockcellulardifferentiationinvivo(Laietal,2012;

Soleimanietal.,2012)potentiallyactingascellularreprogrammingagents.

WechosetoexaminetheclassicalEMTtranscriptionfactorsSnail1andSnail2which

havebothbeenimplicatedinstemcellinductionaswellasanotherEMT

transcriptionfactorZeb2(SIP1)thathasbeensuggestedtodrivecellfatedecisions

(LimandThiery,2012).Surprisingly,weobservedasignificantupregulationof

mRNAencodingSnail1andSnail2inblastematissueatstageIVofregeneration.To

ourknowledge,thisisthefirstdemonstrationofEMT‐associatedzincfinger

transcriptionfactorsSnail1andSnail2beingupregulatedduringappendage

regenerationandsuggeststhatthesetranscriptionfactorscouldbeplaying

importantrolesinregeneration.Otherstudiesusingtheaxolotlhaveshownthatthe

EMTassociatedbasichelixloophelix(bHLH)transcriptionfactorsTwist1and

Twist3areupregulatedbyblastemacellsduringbothlimbandtailregeneration

(Satohetal.,2008;Kragletal,2012).ThesefindingssuggestthatEMT‐associated

transcriptionfactorsareinvolvedinmulti‐tissueregeneration,however,the

63

functionofthesetranscriptionfactorsintheregenerateprogramisyettobe

determined.ItistemptingtospeculatethatSnail1andSnail2arepossibly

functioningindrivingdedifferentiationthatunderliestherapidexpansionofthe

regenerateblastema.Futureworkwillneedtoextensivelycharacterizetheroleof

thesetranscriptionfactorsduringtheregenerateprogramandarelikelytoyield

excitingresults.

64

SummaryandConclusionsNumerouslizardspeciesarecapableofscar‐freewoundhealingandsubsequent

multi‐tissueregenerationfollowinginjuryorlossofappendage(Tanakaand

Reddien,2011).Anemergingmodelforthestudyofscar‐freewoundhealingand

regenerationinlizardsistheleopardgecko(Whimster,1978;McLeanand

Vickaryous,2011;Delormeetal.,2012).Althoughthemolecularregulationofthe

eventsthatunderlytailregenerationintheleopardgeckoremainpoorly

understood,evidencefromvariousotherregeneration‐competentmodelssupports

theinvolvementofvariouscytokinesincludingmembersoftheTGFβsuperfamily.

(Stoick‐Cooperetal.,2007;StocumandCameron,2011).Inparticular,the

TGFβ/activinarmoftheTGFβsuperfamilyhasbeenshowntobeessentialforthe

regenerativeresponseinothermodelsofmulti‐tissueregenerationincluding

zebrafish,AxolotlandXenopus(Jazwinskaetal.,2007;Levesqueetal.,2007;Hoand

Whitman,2008).WiththisinmindwesoughttoinvestigatetheTGFβ/activin

signalingpathwayaswellasdownstreamTGFβtargetgenesatmultiplestages

duringtheleopardgeckoregenerativeprogram.

InthisstudyweobservedarobustinductionofpSMAD2throughoutregenerate

tissuesfromstagesIIItoVIoftailregeneration.pSMAD2wasobservedinthe

nucleuiofnumerouspunitiveblastemacellsasevidencedbyco‐localizationwiththe

proliferationmarkerPCNAsuggestingthatblastemacellsareactivelyrespondingto

TGFβ/activinsignals.Thisresultconfirmsperviousworkinothermodelsystems

thatTGFβ/activinsignalsarehighlyactiveduringmulti‐tissueregeneration.

65

TodeterminewhichTGFβsuperfamilyligandswerecausingthisrobustinductionof

pSMAD2throughoutregenerationweperformedimmunohistochemistryand

westernblottingfortheprototypicalandprofibroticTGFβligand,TGFβ1.

InterestinglyweobservedTGFβ1expressionisabsentfromearlyregeneratetissue

butisobservedinlateregenerationaswellasoriginaltissue.Thisresultindicates

thatTGFβ1islikelynotthemainligandresponsibleforthelargepSMAD2induction

seeninregeneratetissueandsuggeststhatanabsenceofTGFβ1inearlystagesof

tailregenerationmaybenecessaryforsuccessfulscar‐free,multi‐tissue

regeneration.

WethensetouttodetecttheunknownpSMAD2inducingligandandinteresingly

observedthattheonlyTGFβ/activinfamilymembersignificantlyupregulatedin

earlyregeneratetissueswasactivin­βA.Activinligandsarecapableofcausing

SMAD2phosphorylationandhavebeenobservedtobeupregultedatearlystagesof

zebrafishtailregeneration(Jazawinskaetal.,2007).Thisresultssupportsprevious

workinthezebrafishandsuggeststhatactivinligandsareplayinguniqueand

importantrolesinthetissueregenerationprocess.

Wealsoobservedthatinearlystagesoftailregenerationepithelialtomesecnhymal

transitiontranscriptionfactorsaresignificantlyupregulatedinregeneratetissue.

ThissuggeststhattheEMTisactiveduringtailregenerationintheleopardgecko

66

andthatEMT(s)couldbeanimportant,TGFβmediatedcellularprogramsthat

underlieregenerativeability.

TheseresultssupportthepreviouslydescribedimportanceofTGFβ/activin

signalinginnumerousmodelsofmulti‐tissueregeneration.Furthermore,the

observationofdynamicTGFβ1expressionatvariousstagesofregeneration

combinedwiththeobservationofincreasedexpressionofactivin‐βAinregenerate

tissuespositionsTGFβ/activinisoformsasimportantdeterminantsofregenerate

complianceandsuccess.Finally,theupregulationofEMTtranscriptionfactorsin

earlyregeneratetissuessuggestsamodelwherespecificTGFβ/activinisoformscan

activateparticularcellularprograms,suchasEMT,thatcontributetoregeneration

andavoidscarformation.TheseTGFβ/activinsignalingnetworksneedtobefuther

elucidatedbutlikelyholdmuchpromiseforimprovingourunderstandingofthe

factorsthatdrivethebalancebetweenscarformationandtissueregeneration.

67

SignificanceandFutureDirectionsTheresultspresentedhereimproveourcurrentunderstandingofTGFβ/activin

signalingduringscar‐freewoundhealingandmulti‐tissueregeneration.The

demonstrationofrobustTGFβ/activinsignalingintheregeneratetissueofan

amniotecapableofmulti‐tissueregenerationisthefirstofitskindandexpandson

workdemonstratingTGFβ/activinsignalinginanamniotescapabableof

regeneration.Furthermore,thisworkalludestotheimportantrolesofdifferent

TGFβ/activinligands,inparticularTGFβ1andactivin‐βAduringscar‐freewound

healingandmulti‐tissueregeneration.Workexamingtheproteinexpressionof

variousTGFβligandsinothermodelsofmulti‐tissueregenerationhasbeenlacking

fromtheliteratureduetolackofappropriateantibodies.Byexaminingprotein

localizationthroughimmunohistochemistryandwesternblotwewereableto

observethatTGFβ1expressionisabsentfromearlyregeneratetissue,anovel

findingthatisinlinewithotherliteraturesuggestingTGFβ1isprofibroticandthat

itsexpressioncandrivescarification.DespitetheabsenceofTGFβ1ligand

expressionwestillobserverobustpSMAD2nuclearlocalizationincellsofthe

regeneratetissue,suggestingalternateligandsaredrivingSMAD2phosphoryaltion.

Weidentifythatactivin‐βAislikelythepSMAD2activatingligandinregenerate

tissue.Thisresultconfirmsthepreviouslyobservedupregulationofactivin‐βAin

zebrafishtailregenerationandhighlightsthepotentialimportanceofactivinligands

duringscar‐free,multi‐tissueregeneration.Finallywemakethenovelobservation

ofEMTtranscriptionfactorsSnail1andSnail2beingupregulatedinregenerate

68

tissuesuggestingepithelialtomesenchymaltransitionsmaybeimportantfor

regenerativecompliance.

Toexpandonthisworkmoreexperimentsshouldbeconductedexaminingactivin

ligands,thelocalizationofEMTtranscriptionfactorsaswellasgainandlossof

functionstudies.

Activinligandsareformedfromheteroandhomodimerswithdifferentinhibin

subunits,thus,examiningsubunitmRNAexpressionwithqRT‐PCRdoesnotreveal

whatligandsareassembled.Thesedifferentligandshavebeendemonstratedto

havedifferentcellulareffectsandthusclarifyingthedimercompoisitionisan

importantnextstepforthisproject.

ItwillalsobeusefultobettercharacterizetheexpressionofEMTtranscription

factorsSnail1andSnail2duringtissueregeneationusingtechniquesthatlocalize

theirexpression.EMTtranscriptionfactorsarenotoriouslydifficulttolocalizeusing

antibodiesandthustechniquessuchasin‐situhybridizationmayneedtobe

considered.BylocalizingEMTtranscriptionfactorstheidentificationofpotential

cellswithinparticulartissuesthatmaybeundergoingepithelialtomesenchymal

transitionscanbedetermined.Oncelocalized,furtherworkcanbeconductedto

examinewhatrolesEMTtranscriptionfactorshaveinthetissueregeneration

process.

69

Finally,gainandlossoffunctionstudiesneedtobeconductedin‐vivoandin‐vitroto

betterunderstandthefunctionaleffectsofTGFβ/activinligandsduringthe

regenerativeprocessinthegecko.Withthisinmind,usefultechniquescouldbe

mRNAsilencingusingknockdownmorphilinosandsmallRNAmolecules,protein

reductionusingneutralizingantibodies,aswellaspathwayinhibitionusingsmall

moleculeinhibitors.Furthermore,theoverexpressionofdifferentligandscouldbe

accomplishedthroughtheimplantionofagarosebeadssoakedinrecombinant

proteins,adenovirusmediatedoverexpressionorthroughthetreatmentoftissue

explantsin‐vitro.Thesedifferenttechniquescouldbeappliedtovariousresearch

questionsandwouldaidinourunderstandingofthefunctionalconsequencesof

underorover‐expressionofvariousTGFβ/activinligands.

70

ReferencesAlibardiL.2010.Morphologicalandcellularaspectsoftailandlimbregenerationin

lizards.Amodelsystemwithimplicationsfortissueregenerationinmammals.Adv

AnatEmbryolCellBiol207:iii,v‐x,1‐109.

AomatsuK,AraoT,AbeK,KodamaA,SugiokaK,MatsumotoK.2012.Slugis

upregulatedduringwoundhealingandregulatescellularphenotypesincorneal

epithelialcells.InvestOphthalmolVisSci.53:751‐756.

AshcroftGS,YangX,GlickAB,WeinsteinM,LetterioJL,MizelDE,AnzanoM,

Greenwell‐WildT,WahlSM,DengC,RobertsAB.1999.MicelackingSmad3show

acceleratedwoundhealingandanimpairedlocalinflammatoryresponse.NatCell

Biol1:260‐266.

AttisanoL,WranaJL,MontalvoE,MassagueJ.1996.Activationofsignallingbythe

activinreceptorcomplex.MolCellBiol16:1066‐1073.

BellairsA,BryantSV.1985.Autotomyandregenerationinreptiles.In:GansC,Billett

F,editors.Biologyofreptilian.Vol.15.NewYork:WileyandSons.p.303‐410.

BeyerTA,NarimatsuM,WeissA,DavidL,WranaJL.2012.TheTGFbetasuperfamily

instemcellbiologyandearlymammalianembryonicdevelopment.BiochimBiophys

71

Acta.

BielefeldKA,Amini‐NikS,AlmanBA.2012.Cutaneouswoundhealing:recruiting

developmentalpathwaysforregeneration.CellMolLifeSci.

BiernackaA,DobaczewskiM,FrangogiannisNG.2011.TGF‐betasignalingin

fibrosis.GrowthFactors29:196‐202.

BrockesJP.1984.Mitogenicgrowthfactorsandnervedependenceoflimb

regeneration.Science225:1280‐1287.

BrockesJP,KumarA.2002.Plasticityandreprogrammingofdifferentiatedcellsin

amphibianregeneration.NatRevMolCellBiol3:566‐574.

BurgerHG,IgarashiM,BairdDT,BardinW,ChappelS,deJongF,DemoulinA,de

KretserD,FindlayJ,ForageR,etal.1988.Inhibin:definitionandnomenclature,

includingrelatedsubstances.ClinEndocrinol(Oxf)28:448‐449.

BustinSA,BenesV,GarsonJA,HellemansJ,HuggettJ,KubistaM,MuellerR,NolanT,

PfafflMW,ShipleyGL,VandesompeleJ,WittwerCT.2009.TheMIQEguidelines:

minimuminformationforpublicationofquantitativereal‐timePCRexperiments.

ClinChem55:611‐622.

72

CampbellLJ,CrewsCM.2008.Woundepidermisformationandfunctioninurodele

amphibianlimbregeneration.CellMolLifeSci.65:73‐79.

CampbellLJ,Suarez‐CastilloEC,Ortiz‐ZuazagaH,KnappD,TanakaEM,CrewsCM.

2011.Geneexpressionprofileoftheregenerationepitheliumduringaxolotllimb

regeneration.DevDyn.240:1826‐1840.

CarcamoJ,WeisFM,VenturaF,WieserR,WranaJL,AttisanoL,MassagueJ.1994.

TypeIreceptorsspecifygrowth‐inhibitoryandtranscriptionalresponsesto

transforminggrowthfactorbetaandactivin.MolCellBiol14:3810‐3821.

ChristensenRN,TassavaRA.2000.Apicalepithelialcapmorphologyandfibronectin

geneexpressioninregeneratingaxolotllimbs.DevDyn217:216‐224.

ColwellAS,KrummelTM,LongakerMT,LorenzHP.2006.Wnt‐4expressionis

increasedinfibroblastsafterTGF‐beta1stimulationandduringfetalandpostnatal

woundrepair.PlastReconstrSurg117:2297‐2301.

CoolenNA,SchoutenKC,BoekemaBK,MiddelkoopE,UlrichMM.2010.Wound

healinginafetal,adult,andscartissuemodel:acomparativestudy.WoundRepair

Regen18:291‐301.

CordeiroMF,GayJA,KhawPT.1999.Humananti‐transforminggrowthfactor‐beta2

73

antibody:anewglaucomaanti‐scarringagent.InvestOphthalmolVisSci40:2225‐

2234.

CrawfordSE,StellmachV,Murphy‐UllrichJE,RibeiroSM,LawlerJ,HynesRO,Boivin

GP,BouckN.1998.Thrombospondin‐1isamajoractivatorofTGF‐beta1invivo.Cell

93:1159‐1170.

DaCostaByfieldS,MajorC,etal.2004.AB:SB‐505124isaselectiveinhibitorof

transforminggrowthfactorbetatypeIreceptorsALK4,ALK5,andALK7.Mol

Pharmacol,65:744‐752.

DeCrescenzoG,HinckCS,ShuZ,ZunigaJ,YangJ,TangY,BaardsnesJ,MendozaV,

SunL,Lopez‐CasillasF,O'Connor‐McCourtM,HinckAP.2006.Threekeyresidues

underliethedifferentialaffinityoftheTGFbetaisoformsfortheTGFbetatypeII

receptor.JMolBiol355:47‐62.

DelormeSL,LunguIM,VickaryousMK.2012.Scar‐freewoundhealingand

regenerationfollowingtaillossintheleopardgecko,Eublepharismacularius.Anat

Rec(Hoboken)295:1575‐1595.

DennlerS,HuetS,GauthierJM.1999.Ashortamino‐acidsequenceinMH1domainis

responsibleforfunctionaldifferencesbetweenSmad2andSmad3.Oncogene

18:1643‐1648.

74

DerynckR,MiyazonoKo.2007.TheTGF‐[beta]family.ColdSpringHarbor,N.Y.:Cold

SpringHarborLaboratoryPress.

DicksonMC,MartinJS,CousinsFM,KulkarniAB,KarlssonS,AkhurstRJ.1995.

Defectivehaematopoiesisandvasculogenesisintransforminggrowthfactor‐beta1

knockoutmice.Development121:1845‐1854.

DuboisCM,LapriseMH,BlanchetteF,GentryLE,LeducR.1995.Processingof

transforminggrowthfactorbeta1precursorbyhumanfurinconvertase.JBiol

Chem270:10618‐10624.

DzwonekJ,PreobrazhenskaO,CazzolaS,ConidiA,SchellensA,vanDintherM,

StubbsA,KippelA,HuylebroeckD,tenDijkeP,VerschuerenK.2009.Smad3isakey

nonredundantmediatoroftransforminggrowthfactorbetasignalinginNmemouse

mammaryepithelialcells.MolCancerRes7:1342‐1353.

FengXH,DerynckR.2005.Specificityandversatilityintgf‐betasignalingthrough

Smads.AnnuRevCellDevBiol21:659‐693.

FergusonMW,O'KaneS.2004.Scar‐freehealing:fromembryonicmechanismsto

adulttherapeuticintervention.PhilosTransRSocLondBBiolSci359:839‐850.

75

FergusonMW,WhitbyDJ,ShahM,ArmstrongJ,SiebertJW,LongakerMT.1996.Scar

formation:thespectralnatureoffetalandadultwoundrepair.PlastReconstrSurg

97:854‐860.

FernandoWA,LeiningerE,SimkinJ,LiN,MalcomCA,SathyamoorthiS,HanM,

MuneokaK.2011.Woundhealingandblastemaformationinregeneratingdigittips

ofadultmice.DevBiol350:301‐310.

GlobusM,Vethamany‐GlobusS,LeeYC.1980.Effectofapicalepidermalcapon

mitoticcycleandcartilagedifferentiationinregenerationblastematainthenewt,

Notophthalmusviridescens.DevBiol75:358‐372.

GordonKJ,BlobeGC.2008.Roleoftransforminggrowthfactor‐betasuperfamily

signalingpathwaysinhumandisease.BiochimBiophysActa1782:197‐228.

GoumansMJ,ValdimarsdottirG,ItohS,RosendahlA,SiderasP,tenDijkeP.2002.

BalancingtheactivationstateoftheendotheliumviatwodistinctTGF‐betatypeI

receptors.EMBOJ21:1743‐1753.

GrayPC,GreenwaldJ,BlountAL,KunitakeKS,DonaldsonCJ,ChoeS,ValeW.2000.

IdentificationofabindingsiteonthetypeIIactivinreceptorforactivinandinhibin.

JBiolChem275:3206‐3212.

76

GuoW,KeckesovaZ,DonaherJL,ShibueT,TischlerV,ReinhardtF,ItzkovitzS,

NoskeA,Zurrer‐HardiU,BellG,TamWL,ManiSA,vanOudenaardenA,Weinberg

RA.2012.SlugandSox9cooperativelydeterminethemammarystemcellstate.Cell

148:1015‐1028.

GuoX,WangXF.2009.Signalingcross‐talkbetweenTGF‐beta/BMPandother

pathways.CellRes19:71‐88.

GurtnerGC,WernerS,BarrandonY,LongakerMT.2008.Woundrepairand

regeneration.Nature453:314‐321.

HanM,YangX,LeeJ,AllanCH,MuneokaK.2008.Developmentandregenerationof

theneonataldigittipinmice.DevBiol315:125‐135.

HanM,YangX,TaylorG,BurdsalCA,AndersonRA,MuneokaK.2005.Limb

regenerationinhighervertebrates:developingaroadmap.AnatRecBNewAnat

287:14‐24.

HarrisonCA,GrayPC,ValeWW,RobertsonDM.2005.Antagonistsofactivin

signaling:mechanismsandpotentialbiologicalapplications.TrendsEndocrinol

Metab16:73‐78.

Heber‐KatzE.1999.Theregeneratingmouseear.SeminCellDevBiol.4:415‐419.

77

HeldinCH,VanlandewijckM,MoustakasA.2012.RegulationofEMTbyTGFbetain

cancer.FEBSLett.586:1959‐70.

HillC,FlyvbjergA,RaschR,BakM,LoganA.2001.Transforminggrowthfactor‐

beta2antibodyattenuatesfibrosisintheexperimentaldiabeticratkidney.J

Endocrinol170:647‐651.

HinckAP.2012.StructuralstudiesoftheTGF‐betasandtheirreceptors‐insights

intoevolutionoftheTGF‐betasuperfamily.FEBSLett586:1860‐1870.

HoDM,WhitmanM.2008.TGF‐betasignalingisrequiredformultipleprocesses

duringXenopustailregeneration.DevBiol315:203‐216.

HootKE,LighthallJ,HanG,LuSL,LiA,JuW,Kulesz‐MartinM,BottingerE,WangXJ.

2008.Keratinocyte‐specificSmad2ablationresultsinincreasedepithelial‐

mesenchymaltransitionduringskincancerformationandprogression.JClinInvest

118:2722‐2732.

IllingworthCM.1974.Trappedfingersandamputatedfingertipsinchildren.J

PediatrSurg9:853‐858.

InmanGJ,NicolasFJ,CallahanJF,HarlingJD,GasterLM,ReithAD,LapingNJ,HillCS.

2002.SB‐431542isapotentandspecificinhibitoroftransforminggrowthfactor‐

78

betasuperfamilytypeIactivinreceptor‐likekinase(ALK)receptorsALK4,ALK5,

andALK7.MolPharmacol62:65‐74.

JazwinskaA,BadakovR,KeatingMT.2007.Activin‐betaAsignalingisrequiredfor

zebrafishfinregeneration.CurrBiol17:1390‐1395.

JianH,ShenX,LiuI,SemenovM,HeX,WangXF.2006.Smad3‐dependentnuclear

translocationofbeta‐cateninisrequiredforTGF‐beta1‐inducedproliferationof

bonemarrow‐derivedadulthumanmesenchymalstemcells.GenesDev20:666‐674.

JoplingC,BoueS,IzpisuaBelmonteJC.2011.Dedifferentiation,transdifferentiation

andreprogramming:threeroutestoregeneration.NatRevMolCellBiol12:79‐89.

KaartinenV,VonckenJW,ShulerC,WarburtonD,BuD,HeisterkampN,GroffenJ.

1995.AbnormallungdevelopmentandcleftpalateinmicelackingTGF‐beta3

indicatesdefectsofepithelial‐mesenchymalinteraction.NatGenet11:415‐421.

KalluriR,ZeisbergM.2006.FibroblastsinCancer.NatRevCancer6:392‐401.

KangJS,LiuC,DerynckR.2009.NewregulatorymechanismsofTGF‐betareceptor

function.TrendsCellBiol19:385‐394.

KraglM,KnappD,NacuE,KhattakS,MadenM,EpperleinHH,TanakaEM.2009.

79

Cellskeepamemoryoftheirtissueoriginduringaxolotllimbregeneration.Nature

460:60‐65.

KraglM,RoenschK,NussleinI,TazakiA,TaniguchiY,TaruiH,HayashiT,AgataK,

TanakaEM.2012.Muscleandconnectivetissueprogenitorpopulationsshow

distinctTwist1andTwist3expressionprofilesduringaxolotllimbregeneration.Dev

Biol.373:196‐204.

KretschmerA,MoepertK,DamesS,SternbergerM,KaufmannJ,KippelA.2003.

DifferentialregulationofTGF‐betasignalingthroughSmad2,Smad3andSmad4.

Oncogene22:6748‐6763.

LaiSL,MillerMR,RobinsonKJ,DoeCQ.2012.Thesnailfamilymemberworniuis

continuouslyrequiredinneuroblaststopreventelav‐inducedpremature

differentiation.DevCell23:849‐857.

LarsonBJ,LongakerMT,LorenzHP.2010.Scarlessfetalwoundhealing:abasic

sciencereview.PlastReconstrSurg126:1172‐1180.

LavertyHG,WakefieldLM,OcclestonNL,O'KaneS,FergusonMW.2009.TGF‐beta3

andcancer:areview.CytokineGrowthFactorRev20:305‐317.

LeeMK,PardouxC,HallMC,LeePS,WarburtonD,QingJ,SmithSM,DerynckR.

80

2007.TGF‐betaactivatesErkMAPkinasesignallingthroughdirectphosphorylation

ofShcA.EMBOJ26:3957‐3967.

LeferovichJM,Heber‐KatzE.2002.Thescarlessheart.SeminCellDevBiol13:327‐

333.

LehoczkyJA,RobertB,TabinCJ.2011.Mousedigittipregenerationismediatedby

fate‐restrictedprogenitorcells.ProcNatlAcadSciUSA108:20609‐20614.

LeopoldPL,VincentJ,WangH.2012.Acomparisonofepithelialtomesenchymal

transitionandre‐epithelialization.SeminCancerBiol.22:471‐483

LevesqueM,GatienS,FinnsonK,DesmeulesS,VilliardE,PiloteM,PhilipA,RoyS.

2007.Transforminggrowthfactor:betasignalingisessentialforlimbregeneration

inaxolotls.PLoSOne2:e1227.

LewisKA,GrayPC,BlountAL,MacConellLA,WiaterE,BilezikjianLM,ValeW.2000.

Betaglycanbindsinhibinandcanmediatefunctionalantagonismofactivin

signalling.Nature404:411‐414.

LimJ,ThieryJP.2012.Epithelial‐mesenchymaltransitions:insightsfrom

development.Development139:3471‐3486.

81

LinRY,AdzickNS.1996.Theroleofthefetalfibroblastandtransforminggrowth

factor‐betainamodelofhumanfetalwoundrepair.SeminPediatrSurg5:165‐174.

LiuK,JohnsonK,LiJ,PiamonteV,SteffyBM,HsiehMH,NgN,ZhangJ,etal.2011.

Regenerativephenotypeinmicewithapointmutationintransforminggrowth

factorbetatype1receptor(TGFBR1).ProcNatlAcadSci.108.14560‐14565.

MacConellLA,LealAM,ValeWW.2002.Thedistributionofbetaglycanproteinand

mRNAinratbrain,pituitary,andgonads:implicationsforaroleforbetaglycanin

inhibin‐mediatedreproductivefunctions.Endocrinology143:1066‐1075.

ManiSA,GuoW,LiaoMJ,EatonEN,AyyananA,ZhouAY,BrooksM,ReinhardF,

ZhangCC,ShipitsinM,CampbellLL,PolyakK,BriskenC,YangJ,WeinbergRA.2008.

Theepithelial‐mesenchymaltransitiongeneratescellswithpropertiesofstemcells.

Cell133:704‐715.

MassagueJ.2008.TGFbetainCancer.Cell134:215‐230.

MartinP.1997.Woundhealng–aimingforperfectskinregeneration.Science.

276:75‐81

MatzukMM,KumarTR,BradleyA.1995.Differentphenotypesformicedeficientin

eitheractivinsoractivinreceptortypeII.Nature374:356‐360.

82

McDowallM,EdwardsNM,JahodaCA,HyndPI.2008.Theroleofactivinsand

follistatinsinskinandhairfollicledevelopmentandfunction.CytokineGrowth

FactorRev19:415‐426.

McLeanKE,VickaryousMK.2011.Anovelamniotemodelofepimorphic

regeneration:theleopardgecko,Eublepharismacularius.BMCDevBiol11:50.

MonaghanJR,AthippozhyA,SeifertAW,PuttaS,StrombergAJ,MadenM,Gardiner

DM,VossSR.2012.Geneexpressionpatternsspecifictotheregeneratinglimbofthe

Mexicanaxolotl.BiolOpen1:937‐948.

MoustakasA,HeldinCH.2005.Non‐SmadTGF‐betasignals.JCellSci118:3573‐

3584.

MoustakasA,HeldinCH.2009.TheregulationofTGFbetasignaltransduction.

Development136:3699‐3714.

MuneokaK,AllanCH,YangX,LeeJ,HanM.2008.Mammalianregenerationand

regenerativemedicine.BirthDefectsResCEmbryoToday84:265‐280.

MungerJS,SheppardD.2011.CrosstalkamongTGF‐betasignalingpathways,

integrins,andtheextracellularmatrix.ColdSpringHarbPerspectBiol3:a005017.

83

NacuE,TanakaEM.2011.Limbregeneration:anewdevelopment?AnnuRevCell

DevBiol27:409‐440.

NawshadA,LaGambaD,HayED.2004.Transforminggrowthfactorbeta(TGFbeta)

signallinginpalatalgrowth,apoptosisandepithelialmesenchymaltransformation

(EMT).ArchOralBiol49:675‐689.

NayaDE,VelosoC,MunozJL,BozinovicF.2007.Somevaguelyexplored(butnot

trivial)costsoftailautotomyinlizards.CompBiochemPhysiolAMolIntegrPhysiol

146:189‐193.

NeufeldDA,DayFA.1996.Perspective:asuggestedroleforbasementmembrane

structuresduringnewtlimbregeneration.AnatRec246:155‐161.

NietoMA.2011.Theinsandoutsoftheepithelialtomesenchymaltransitionin

healthanddisease.AnnuRevCellDevBiol.27:347‐376

OcclestonNL,O'KaneS,LavertyHG,CooperM,FairlambD,MasonT,BushJA,

FergusonMW.2011.Discoveryanddevelopmentofavotermin(recombinanthuman

transforminggrowthfactorbeta3):anewclassofprophylactictherapeuticforthe

improvementofscarring.WoundRepairRegen19Suppl1:s38‐48.

84

OgunjimiAA,ZeqirajE,CeccarelliDF,SicheriF,WranaJL,DavidL.2012.Structural

basisforspecificityofTGFbetafamilyreceptorsmallmoleculeinhibitors.CellSignal

24:476‐483.

OshimoriN,FuchsE.2012.TheharmoniesplayedbyTGF‐betainstemcellbiology.

CellStemCell11:751‐764.

OzdamarB,BoseR,Barrios‐RodilesM,WangHR,ZhangY,WranaJL.2005.

RegulationofthepolarityproteinPar6byTGFbetareceptorscontrolsepithelialcell

plasticity.Science307:1603‐1609.

PetersenM,PardaliE,vanderHorstG,CheungH,vandenHoogenC,vanderPluijm

G,TenDijkeP.2010.Smad2andSmad3haveopposingrolesinbreastcancerbone

metastasisbydifferentiallyaffectingtumorangiogenesis.Oncogene29:1351‐1361.

PeinadoH,OlmedaD,CanoA.2007.Snail,Zeb,andbHLHfactorsintumor

progression:anallianceagainsttheepithelialphenotype.7:415‐428.

PossKD.2007.Gettingtotheheartofregenerationinzebrafish.SeminCellDevBiol

18:36‐45.

PossKD.2010.Advancesinunderstandingtissueregenerativecapacityand

mechanismsinanimals.NatRevGenet11:710‐722.

85

ProetzelG,PawlowskiSA,WilesMV,YinM,BoivinGP,HowlesPN,DingJ,Ferguson

MW,DoetschmanT.1995.Transforminggrowthfactor‐beta3isrequiredfor

secondarypalatefusion.NatGenet11:409‐414.

QuagginSE,KapusA.2011.Scarwars:mappingthefateoftheepithelial

mesenchymalmyofibroblasttransition.2011.KidenyInt.80:41‐50.

RajnochC,FergusonS,MetcalfeAD,HerrickSE,WillisHS,FergusonMW.2003.

Regenerationoftheearafterwoundingindifferentmousestrainsisdependenton

theseverityofwoundtrauma.DevDyn226:388‐397.

ReinkeJM,SorgH.2012.Woundrepairandregeneration.EurSurgRes49:35‐43.

RifkinDB.2005.Latenttransforminggrowthfactor‐beta(TGF‐beta)binding

proteins:orchestratorsofTGF‐betaavailability.JBiolChem280:7409‐7412.

RinkevichY,LindauP,UenoH,LongakerMT,WeissmanIL.2011.Germ‐layerand

lineage‐restrictedstem/progenitorsregeneratethemousedigittip.Nature

476:409‐413.

RossS,HillCS.2008.HowtheSmadsregulatetranscription.IntJBiochemCellBiol

40:383‐408.

86

RowlattU.1979.Intrauterinewoundhealingina20weekhumanfetus.Virchows

ArchAPatholAnatHistol381:353‐361.

SanchezAlvaradoA.2006.Planarianregeneration:itsendisitsbeginning.Cell

124:241‐245.

SandhuC,GarbeJ,BhattacharyaN,DaksisJ,PanCH,YaswenP,KohJ,SlingerlandJM,

StampferMR.1997.Transforminggrowthfactorbetastavilizesp15INK4Bprotein,

increasesp15INK4B‐cdk4complexes,andinhibitscyclinD1‐cdk4associationin

humanmammaryepithelialcells.MolCellBiol17:2458‐2467.

SanfordLP,OrmsbyI,Gittenberger‐deGrootAC,SariolaH,FriedmanR,BoivinGP,

CardellEL,DoetschmanT.1997.TGFbeta2knockoutmicehavemultiple

developmentaldefectsthatarenon‐overlappingwithotherTGFbetaknockout

phenotypes.Development124:2659‐2670.

SatohA,BryantSV,GardinerDM.2008.Regulationofdermalfibroblast

dedifferentiationandredifferentiationduringwoundhealingandlimbregeneration

intheAxolotl.DevGrowthDiffer50:743‐754.

SavangerP,Arnoux.2009.Epithelio‐mesenchymaltransitionandcutaneouswound

healing.BullAcadNatlMed.193:1981‐1991.

87

ScheelC,EatonEN,LiSH,ChafferCL,ReinhardtF,KahKJ,BellG,GuoW,RubinJ,

RichardsonAL,WeinbergRA.2011.Paracrineandautocrinesignalsinduceand

maintainmesenchymalandstemcellstatesinthebreast.Cell.145:926‐940.

SchmiererB,SchusterMK,ShkumatavaA,KuchlerK.2003.Activinasignaling

inducesSmad2,butnotSmad3,requiringproteinkinaseaactivityingranulosacells

fromtheavianovary.JBiolChem278:21197‐21203.

SeifertAW,KiamaSG,SeifertMG,GoheenJR,PalmerTM,MadenM.2012.Skin

sheddingandtissueregenerationinAfricanspinymice(Acomys).Nature489:561‐

565.

ShahM,ForemanDM,FergusonMW.1995.NeutralisationofTGF‐beta1andTGF‐

beta2orexogenousadditionofTGF‐beta3tocutaneousratwoundsreduces

scarring.JCellSci108(Pt3):985‐1002.

SheppardD.2005.Integrin‐mediatedactivationoflatenttransforminggrowthfactor

beta.CancerMetastasisRev24:395‐402.

ShiY,MassagueJ.2003.MechanismsofTGF‐betasignalingfromcellmembraneto

thenucleus.Cell113:685‐700.

88

ShullMM,OrmsbyI,KierAB,PawlowskiS,DieboldRJ,YinM,AllenR,SidmanC,

ProetzelG,CalvinD,etal.1992.Targeteddisruptionofthemousetransforming

growthfactor‐beta1generesultsinmultifocalinflammatorydisease.Nature

359:693‐699.

SoleimaniVD,YinH,Jahani‐AslA,MingH,KockxCE,vanIjckenWF,GrosveldF,

RudnickiMA.2012.SnailregulatesMyoDbinding‐siteoccupancytodirectenhancer

switchinganddifferentiation‐specifictranscriptioninmyogenesis.MolCell47:457‐

468.

SongF,LiB,StocumDL.2010.Amphibiansasresearchmodelsforregenerative

medicine.Organogenesis6:141‐150.

SooC,BeanesSR,HuFY,ZhangX,DangC,ChangG,WangY,NishimuraI,Freymiller

E,LongakerMT,LorenzHP,TingK.2003.Ontogenetictransitioninfetalwound

transforminggrowthfactor‐betaregulationcorrelateswithcollagenorganization.

AmJPathol163:2459‐2476.

SorrentinoA,ThakurN,GrimsbyS,MarcussonA,vonBulowV,SchusterN,ZhangS,

HeldinCH,LandstromM.2008.ThetypeITGF‐betareceptorengagesTRAF6to

activateTAK1inareceptorkinase‐independentmanner.NatCellBiol10:1199‐

1207.

89

SouchelnytskyiS,tenDijkeP,MiyazonoK,HeldinCH.1996.Phosphorylationof

Ser165inTGF‐betatypeIreceptormodulatesTGF‐beta1‐inducedcellular

responses.EMBOJ15:6231‐6240.

StocumDL,CameronJA.2011.Lookingproximallyanddistally:100yearsoflimb

regenerationandbeyond.DevDyn240:943‐968.

Stoick‐CooperCL,MoonRT,WeidingerG.2007.Advancesinsignalinginvertebrate

regenerationasapreludetoregenerativemedicine.GenesDev21:1292‐1315.

TalTL,FranzosaJA,TanguayRL.2010.Molecularsignalingnetworksthat

choreographepimorphicfinregenerationinzebrafish‐amini‐review.Gerontology

56:231‐240.

TamuraK,OhgoS,YokoyamaH.2010.Limbblastemacell:astemcellfor

morphologicalregeneration.DevGrowthDiffer52:89‐99.

TanakaEM.ReddienPW.2011.Thecellularbasisforanimalregeneration.DevCell.

21:172‐185.

TaylorS,WakemM,DijkmanG,AlsarrajM,NguyenM.2010.Apracticalapproachto

RT‐qPCR‐PublishingdatathatconformtotheMIQEguidelines.Methods50:S1‐5.

90

tenDijkeP,ArthurHM.2007.ExtracellularcontrolofTGFbetasignallinginvascular

developmentanddisease.NatRevMolCellBiol8:857‐869.

ThieryJP.2002.Epithelial‐mesenchymaltransitionsintumourprogression.NatRev

Cancer2:442‐454.

ThieryJP,AcloqueH,HuangRY,NietoMA.2009.Epithelial‐mesencymaltransitions

indevelopmentanddisease.Cell.139:871‐890

ThorntonCS.1957.Theeffectofapicalcapremovalonlimbregenerationin

Amblystomalarvae.JExpZool134:357‐381.

TsengAS,LevinM.2008.TailregenerationinXenopuslaevisasamodelfor

understandingtissuerepair.JDentRes87:806‐816.

TsonisPA,DelRio‐TsonisK,WallaceJL,BurnsJC,HofmannMC,MillanJL,

WashabaughCH.1996.Caninsightsintourodelelimbregenerationbeachieved

withcellculturesandretroviruses?IntJDevBiol40:813‐816.

ValeW,RivierC,HsuehA,CampenC,MeunierH,BicsakT,etal.1988.Chemicaland

biologicalcharacterizationoftheinhibinfamilyofproteinhormones.RecentProg

HormRes.44:1‐44

91

VidalP,DicksonMG.1993.Regenerationofthedistalphalanx.Acasereport.JHand

SurgBr18:230‐233.

VittUA,HsuSY,HsuehAJ.2001.Evolutionandclassificationofcystineknot‐

containinghormonesandrelatedextracellularsignalingmolecules.MolEndocrinol

15:681‐694.

WiaterE,ValeW.2008.SignalingreceptorsoftheTGF‐βfamily.In:DerynckR,

MiyazonoK,editors.TheTGF‐βFamily.ColdSpringHarbor:ColdSpringHarbor

LaboratoryPress.p.151‐177

WallaceH,WatsonA,EgarM.1981.Regenerationofsubnormallyinnervatedaxolotl

arms.JEmbryolExpMorphol62:1‐11.

WeinsteinM,YangX,LiC,XuX,GotayJ,DengCX.1998.Failureofeggcylinder

elongationandmesoderminductioninmouseembryoslackingthetumor

suppressorsmad2.ProcNatlAcadSciUSA95:9378‐9383.

WernerS,AlzheimerC.2006.Rolesofactivinintissuerepair,fibrosis,and

inflammatorydisease.CytokineGrowthFactorRev17:157‐171.

WhimsterIW.1978.Nervesupplyasastimulatorofthegrowthoftissueincluding

skin.II.Animalevidence.ClinExpDermatol3:389‐412.

92

WhitbyDJ,FergusonMW.1991.Immunohistochemicallocalizationofgrowth

factorsinfetalwoundhealing.DevBiol147:207‐215.

WranaJL,AttisanoL,WieserR,VenturaF,MassagueJ.1994.Mechanismof

activationoftheTGF‐betareceptor.Nature370:341‐347.

WranaJL,OzdamarB,LeRoyC,BenchabaneH.2008.SignalingreceptorsoftheTGF‐

βfamily.In:DerynckR,MiyazonoK,editors.TheTGF‐βFamily.ColdSpringHarbor:

ColdSpringHarborLaboratoryPress.p.151‐177

WuL,SiddiquiA,MorrisDE,CoxDA,RothSI,MustoeTA.1997.Transforming

growthfactorbeta3(TGFbeta3)accelerateswoundhealingwithoutalterationof

scarprominence.Histologicandcompetitivereverse‐transcription‐polymerase

chainreactionstudies.ArchSurg132:753‐760.

WuMY.HillCS.2009.Tgf‐betasuperfamilysignalinginembryonicdevelopmentand

homeostasis.DevCell.16:329‐343.

WynnTA,RamalingamTR.2012.Mechanismsoffibrosis:therapeutictranslationfor

fibroticdisease.NatMed18:1028‐1040.

XuP,LiuJ,DerynckR.2012.Post‐translationalregulationofTGF‐betareceptorand

93

Smadsignaling.FEBSLett586:1871‐1884.

YamashitaH,tenDijkeP,FranzenP,MiyazonoK,HeldinCH.1994.Formationof

hetero‐oligomericcomplexesoftypeIandtypeIIreceptorsfortransforminggrowth

factor‐beta.JBiolChem269:20172‐20178.

YamashitaM,FatyolK,JinC,WangX,LiuZ,ZhangYE.2008.TRAF6mediatesSmad‐

independentactivationofJNKandp38byTGF‐beta.MolCell31:918‐924.

YangX,LetterioJJ,LechleiderRJ,ChenL,HaymanR,GuH,RobertsAB,DengC.1999.

TargeteddisruptionofSMAD3resultsinimpairedmucosalimmunityand

diminishedTcellresponsivenesstoTGF‐beta.EMBOJ18:1280‐1291.

YangZ,MuZ,DabovicB,JurukovskiV,YuD,SungJ,XiongX,MungerJS.2007.

Absenceofintegrin‐mediatedTGFbeta1activationinvivorecapitulatesthe

phenotypeofTGFbeta1‐nullmice.JCellBiol176:787‐793.

ZeisbergEM,TarnavskiO,ZeisbergM,Dorfman,AL,McMullenJR,etal.2007.

Endothelialtomesenchymaltransitioncontributestocardiacfibrosis.NatMed.

13:952‐961.

ZeisbergEM,PotentaSE,SugimotoH,ZeisbergM,KalluriR.2008.Fibroblastsin

kidneyfibrosisemergeviaendothelialtomesenchymaltransition.JAmSocNephrol.

94

19:2282‐2287.

ZhangY.2009.Non‐SmadpathwaysinTGF‐betasignaling.CellRes19:128‐139

95

AppendixI–PreparationofMaterials7.5%AcrylamideSDS­PAGE2X1.5mmGelsResolvingGel:11 ml distilled water 3.72 ml 40% acrylamide-bis solution 5 ml Tris buffer (1.5 M, pH 8.8) 200 µl 10% SDS 10 µl TEMED 240 µl 10% APS (10 g APS dissolved in 100 ml distilled water) 10%AcrylamideSDS­PAGE2X1.5mmGelsResolvingGel:9.7 ml distilled water 5 ml 40% acrylamide-bis solution 5 ml Tris buffer (1.5 M, pH 8.8) 200 µl 10% SDS 10 µl TEMED 240 µl 10% APS 12%AcrylamideSDS­PAGE2X1.5mmGelsResolvingGel:8.7 ml distilled water 6 ml 40% acrylamide-bis solution 5 ml Tris buffer (1.5 M, pH 8.8) 200 µl 10% SDS 10 µl TEMED 240 µl 10% APS Onceresolvinggelispolymerized,addthestackinggel:3.2mldistilled water 500 µl 40% acrylamide-bis solution 1.26 ml Tris buffer (0.5 M, pH 6.8) 50 µl 10% SDS 5 µl TEMED 50 µl 10% APS CitrateBufferStockSolutionA(0.1MCitricAcid):1.92gramscitricacidpowderin100mldeionizedH2OStockSolutionB(0.1MSodiumCitratedihydrate):14.7gramssodiumcitratedihydratepowderin500mldeionizedH2OMix9.0mLSolutionAwith41mLSolutionB.AdjustpHto6.0.Topupto500mLwithdeionizedH2O,Storeat4°CElectrophoresisBuffer

96

30.25gTris‐base144.1gGlycine100ml10%SDSDissolveinwatertoavolumeof~800ml.AdjustpHto8.3.Adjustfinalvolumeto1L.StoreatroomtemperatureIFBuffer0.1%BSA0.2%Triton‐X0.05%Tween‐20DissolvedinPBS.Storeat4°C.8XProteinLoadingBuffer400mMTris‐HCl(pH6.8)16%SDS0.8%Bromophenolblue40%Glycerol0.4MDTTStoreat­20,oncethawed,storeinthedarkatRTSemi­dryTransferBuffer(1L)5.82gTris2.93gGlycine200mLMethanolDissolveinwatertoafinalvolumeof1L.StoreatRT.TBS(10X)24.2gTris‐base80gNaClDissolveinwatertoavolumeof800ml.AdjustpHto7.6.Adjustfinalvolumeto1L.Storeatroomtemperature.TowbinSolution30.25gTris‐base144.1gGlycineDissolveinwatertoafinalvolumeof1L.Storeat4°C.TransferBuffer100mlTowbinsolution200mlMethanol2.5ml10%SDSAdjustvolumeto1Lwithwater.Chillat4°Canduseimmediately.

97

AppendixII–ChemicalListandSuppliersAurumTotalIsolationRNAkit Bio‐Rad,Hercules,CAAcrylamide/Bissolution(40%) Bio‐Rad,Hercules,CA AlexaFluordonkeyanti‐rabbit488IgG Invitrogen,Camarillo,CAα-Tubulin mouse mAb (B-5-1-2) Sigma‐Aldrich,St.Louis,MOAprotinin Sigma‐Aldrich,St.Louis,MOAPS Bio‐Rad,Hercules,CABromophenolblue Sigma‐Aldrich,St.Louis,MOBSA SantaCruzBio,SantaCruz,CACy3Goatanti‐Mouse JacksonLabs,WestGrove,PADABPeroxidaseSubstrateKit VectorLabs,Burlingame,CADCproteinassay Bio‐Rad,Hercules,CADTT FisherScientific,Nepean,ONGFXPCRGelBandPurificationKit GEHealthcare,Cooksville,ONGlycerol FisherScientific,Nepean,ONGlycine FisherScientific,Nepean,ONGoatanti‐rabbitBiotinylated VectorLabs,Burlingame,CA Goatanti‐mouseHRP Sigma‐Aldrich,St.Louis,MOGoatanti‐rabbitHRP Sigma‐Aldrich,St.Louis,MONa3VO4 NewEnglandBiolabs,Ipswich,MAPBS Lonza,Walkersville,MDPCNAmousemAb(PC‐10) Dako,Burlington,ONPhosphataseInhibitorCocktail2 Sigma‐Aldrich,St.Louis,MOPhospho‐Smad2rabbitpAb(#3101) CellSignaling,Danvers,MAPMSF Sigma‐Aldrich,St.Louis,MOReblotPlus(Mild) Millipore,Billerica,MASmad2mousemAb(#3103) CellSignaling,Danvers,MASDS FisherScientific,Nepean,ONSybrGreenI AppliedBiosystemsTEMED Sigma‐Aldrich,St.Louis,MOTGFβ1(V)rabbitpAb(sc‐146) SantaCruzBio,SantaCruz,CATris‐base FisherScientific,Nepean,ON Trisbuffer(1.5M) Bio‐Rad,Hercules,CATrisbuffer(0.5M) Bio‐Rad,Hercules,CATriton‐X Bio‐Rad,Hercules,CATween‐20 FisherScientific,Nepean,ON VectastainEliteABC(Universal)R.T.U VectorLabs,Burlingame,CA

98

AppendixIII–PrimersandClonedSequenceAnalysisTable1PrimersusedforqRT­PCRPrimer ForwardPrimer ReversePrimerTGFβ1 5‐TCGAGTCATGACAGCTACGG‐3 5‐GGTACAGCAGGTCCGGATTA‐3TGFβ2 5‐CACAATCCAGCGCCTATTTT‐3 5‐TGGCGAAGTTAGGTCTTTGC‐3TGFβ3 5‐AACGCATTGAGCTTTTCCAG‐3 5‐CGCACGGTATCTGTGACATC‐3ActivinβA 5‐CCTGGCACATCTTTCCAGTT‐3 5‐GCTGGCTCCAGTTTCTTGAC‐3Snail1 5‐GCAAGAAGCCCAACTACAGC‐3 5‐AGCCCTGTGTCCCATACAAG‐3Snail2 5‐ATGAGAGCTACCCAATGCCTG‐

35‐CCAAAGATGAGGGATTATCCAGAGA‐3

Zeb2 5‐TCTCAGCCAGAGGAACAAGG‐3 5‐GCTCTGGAGACAAGCTTTGG‐318s 5‐CGGCTACCACATCCTATGAA‐3 5‐TGGAGCTGGAATTACCGCGG‐3

99

Table2PrimersusedforcDNAidentificationPrimer ForwardPrimer ReversePrimer

TGFβ1 5‐AAGCGATCGAAGCCATC‐3 5‐CGYACRCARCAGTTCTYCTC‐3TGFβ2 5‐

ATGCGCAAGAGGATCGAGGCSAT‐3

5‐GGTTCRTGDATCCAYTTCCA‐3

TGFβ3 5‐GCBYTBTACAACAGCACC‐3 5‐CGYACRCARCAGTTCTYCTC‐3ActivinβA 5‐AGGACAAAAGTGACCATCCG‐3 5‐GCTGGCTCCAGTTTCTTGAC‐318s 5‐CGGCTACCACATCCTATGAA‐3 5‐TGGAGCTGGAATTACCGCGG‐3Table3cDNAsequenceanalysis cDNAsequencemaxidentity%with

Gene

cDNA(bp)

Anoliscarolinensis

Gallusgallus

HomoSapiens

GenBankaccession

TGFβ1 839 71 72 83 **TGFβ2 881 81 82 78 **TGFβ3 749 85 83 77 **ActivinβA* 574 87 83 76 JN880655.118s 142 99 99 99 ***ActivinβAwasidentifiedandpublishedduringtheproductionofthismanuscriptbyanothergroup.**CurrentlysubmittedtoGenBank/NCBIawaitingaccessionnumbers

100

AppendixIV–ClonedsequencesActivinβAGGTAGTGGTTGATAACTGTGGAGTGGAAAGAAAGGGTTGATCCAGATGTCCCCGCAATATGGGTGGGGCAGTCTCCTTCACAGTAGTTGGCATGGTATCCTGGTGGGGCAATGATCCAGTCATTCCAGCCAATGTCTTTGAAGCTGACATAGAAGTGCTTCTTGCAGCAGATGTTGACTTTGCCATCGCACTCAAGACCCCGCCTCCGCCGTCTGTGCTGGCGATCCTCTGAATGTCTGGCTAGCATCATCAGGAAGGGCCTATGTGATTGTTCCTTCTCCTCGTCTCCTGTATTTTCACCACCTTCCTTCTCCCTCCCTTCTACGTCTTCTTCCTTTTTTTTCCTCTTACCCAGCAACACCAAGCTGGCTCCAGTTTCTTGACACTGATCACAAGCAATCCGCACATCCAGGGAATTCTTTCCCTGATCCAGAAGGCGTTGAACACTGCTAGAAACTGGAAAGATGTGCCAGGTACTTTTGCGAGTGTCGACTGCCTTTTCAGAGATAAGCATTTCACCTTTTTCTCCCCTCATCCCTCCATCNTCTTCCATTCCTTGGGAATGCGCTTGACATGFβ1CCAGATCCTTAGTAAGCTCAAGCTGTCCAGTCCGCCNGAAAGTGGAAGAGGCCGCGACTTTGTCTGATGAGATTGTGGCACTTTACAATATCACCANGGATTTGATCAAGGAAAAGGTCANGGAGGAGCCACGGGAGACTCCCCAAGAAGAATATTACGCCAAGGAAGTTCACATGTTCAAGATGCTGTCTTCGAGTCATGACAGCTACGGAACACAGTACAAGAGGAACAGCCACAGTATCTTCTTCGCATTTGACCTGGAACAGATCCGGGATGCCCTCCGTAATCCGGACCTGCTGTACCGCGCCGAGCTGAGAATGCTGCGTGGTTCAGACTGGGATCGGTGACTGTGCAGCAGCGGGTGGAGCTTTATCAGCAACAGCAGTTCTCTAGCAACACCTCCTTCTGGGAATACCTGGATGGGCGCACAGTCACTGTGACAGCAGAAAAGGAATGGCTTTCATTTGATGTCACCACTGTCCTTCGCCTCTGGCTTGGGAGCACTGAAACTTTCGGCAGGTTCCGTCTCAGCTCCCAGTGTTCCTGCGGAGAAACCGCTGGAGAACTGAAAGTGGACATAGAAGGCACCAAATCAAAGCGCGGAGACCAGGAGAGCCTCAGCCGCACTCAGAAAGCGCCCTACCTGCTCGTGATGGCGATGCCGCCCGAGCGCGCGGACCAGCTGCAAAGCCACCGGCGTAAGCGAGCCACGCCAGGCATCCAATACTGCAGCGATTCAGCAGAGAAGAACTGTTGCGTGCAGCCTCTTTATATTGACTTCCGTAAGGATTTGAACTGGAAATGGATCCACAAGCCCAGCGGCTACTATGCCAACTTCTGFβ2GGAGCTCTCCNNTATGGTCGACCTGCAGGCGGCCGCGAATTCACTAGTGATTATGCGCAAGAGGATCGAGGCCATCCGAGGGCAGATCCTAAGCAAACTGAACTCACCAGCCCTCCGGAAGAGTTCCCGGAGCCCGAGGAGGTTCCTCCGGAGGTCATTTCAATCTACAACAGCACAAGGGACCTGCTGCAGGAGAAAGCGAACCACAGGGCGGCCACTTGCGAAAGGGAGCGGAGCGAAGAGGAATATTACGCCAAGAGGTTTACAAAATTGATCTGCCTTTTTTCCATTCCGAAAACAAGATTCCACAAAAATCACCACAATCCAGCGCCTATTTTAAACGAGTGAGATTTGACGTCTCAGCAATGGAGAAAAATGCTTCCAACCTCGTGAAGGCAGAATTTAGAGTCTTTCGCTTGCAGAATCCAAAGGCTCGGGTATCAGAACAGCGGATAGAATTATATCAGATTCTCAAAAGCAAAGACCTAACTTCGCCAATGCAACGTTACATTGATAGCAAGGTAGTTAAAACACGA

101

GCCGAAGGTGAATGGCTATCTTTTGATGTAACTGATGCTGTACATGAATGGCTCCATCATAGAGACAGGAATCTGGGATTTAAGATAAGTTTACATTGTCCATGCTGCACGTTCGTGCCTTCAAATAATTATATCATCCCAAACCAAAGTGAAGTGCTAGAAGCAAGTTTTGCAGGNATTGAGGACTACCACAGTGCTGAGAAAACTTTAAAGTCCAATNNNAAAAATACAGNGGGAAGACCCACATCTTCTGCTAATGTTGCTACCCTCCTACAGACTTGAGTCGCANCAGTCCAGTCNANGGANNANCGTGCTTTANATGCTGCCTNTGTTTAGNATGNGNANANACTGCTGNCTGCNACCATTTNCATTGATTCANANGGNNNNTGGNNGNAATGTGFβ3TTCGTACGCAGCAGTTCTTCTCCAGGTTTCGAAAGCAGTAATTGGTGTCTAGTGCTCGTTTTTTCCTTTGGCTTCTTACAGCTGGGCTCCCAAGCCTATGTGGGGGCAGCATCATCAAAATCAAGTGGGGATTGTGAAGGTCTTTCTGCTTCTTCAGGCTCCCTAAGTCCCCACGCCCATGGCTATCCTCGCTGTCGATGCCTTTGAACTTGATCTCCAGCACCTCATGCAGGTTCTCCAAGATGTCACCATTCGACTGGAAGGTATTACATGGGCAGTGGATGCTGATCTCCAGTCCCTGGTTAGATTCTCTATGCAAAAGCCATTCGCGCACGGTATCTGTGACATCAAAGGACAGCCACTCAGCGGAGCCCCGGGTCATCACGTTGCGGCCACTGAGGTAACGCTGCTTTGCAATATGCTCGTCGGGCTTAAGAATCTGGAAAAGCTCAATGCGTTGCTCGGTGCGTTTGGAACTGGGGTTTGGCACTCGCAAGACACGAAATTCAGCCCGGAAGAGGTTGGTGCTGTTCTTTTCTGCTGAAGACACGTTGAAGCGGAACACGTTGGAAGTTACCCCTTTGGGGCAGAATGTCAACTCGTTGTGCTCCGGGAGGCCCTGGATCATGTCAAATTTGTGGATCTCCTTGGCGTAGTACTCGGACTCGGTATTGTCCTGGGAGCAGCTCTCCTCCTTCTCGTCCTCCATCTCCTCCAGAAGCTCCCGGGTGCTGTTGTAAAACGCAATC18sACCCGGGGAGGTAGTGACGAAAAATAACAATACAGGACTCTTTCGAGGCCCTGTAATTGGAATGAGTACACTTTAAATCCTTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAA