characterization of tgfβ signaling during epimorphic
TRANSCRIPT
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.
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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
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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
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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
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AppendixIV–ClonedsequencesActivinβAGGTAGTGGTTGATAACTGTGGAGTGGAAAGAAAGGGTTGATCCAGATGTCCCCGCAATATGGGTGGGGCAGTCTCCTTCACAGTAGTTGGCATGGTATCCTGGTGGGGCAATGATCCAGTCATTCCAGCCAATGTCTTTGAAGCTGACATAGAAGTGCTTCTTGCAGCAGATGTTGACTTTGCCATCGCACTCAAGACCCCGCCTCCGCCGTCTGTGCTGGCGATCCTCTGAATGTCTGGCTAGCATCATCAGGAAGGGCCTATGTGATTGTTCCTTCTCCTCGTCTCCTGTATTTTCACCACCTTCCTTCTCCCTCCCTTCTACGTCTTCTTCCTTTTTTTTCCTCTTACCCAGCAACACCAAGCTGGCTCCAGTTTCTTGACACTGATCACAAGCAATCCGCACATCCAGGGAATTCTTTCCCTGATCCAGAAGGCGTTGAACACTGCTAGAAACTGGAAAGATGTGCCAGGTACTTTTGCGAGTGTCGACTGCCTTTTCAGAGATAAGCATTTCACCTTTTTCTCCCCTCATCCCTCCATCNTCTTCCATTCCTTGGGAATGCGCTTGACATGFβ1CCAGATCCTTAGTAAGCTCAAGCTGTCCAGTCCGCCNGAAAGTGGAAGAGGCCGCGACTTTGTCTGATGAGATTGTGGCACTTTACAATATCACCANGGATTTGATCAAGGAAAAGGTCANGGAGGAGCCACGGGAGACTCCCCAAGAAGAATATTACGCCAAGGAAGTTCACATGTTCAAGATGCTGTCTTCGAGTCATGACAGCTACGGAACACAGTACAAGAGGAACAGCCACAGTATCTTCTTCGCATTTGACCTGGAACAGATCCGGGATGCCCTCCGTAATCCGGACCTGCTGTACCGCGCCGAGCTGAGAATGCTGCGTGGTTCAGACTGGGATCGGTGACTGTGCAGCAGCGGGTGGAGCTTTATCAGCAACAGCAGTTCTCTAGCAACACCTCCTTCTGGGAATACCTGGATGGGCGCACAGTCACTGTGACAGCAGAAAAGGAATGGCTTTCATTTGATGTCACCACTGTCCTTCGCCTCTGGCTTGGGAGCACTGAAACTTTCGGCAGGTTCCGTCTCAGCTCCCAGTGTTCCTGCGGAGAAACCGCTGGAGAACTGAAAGTGGACATAGAAGGCACCAAATCAAAGCGCGGAGACCAGGAGAGCCTCAGCCGCACTCAGAAAGCGCCCTACCTGCTCGTGATGGCGATGCCGCCCGAGCGCGCGGACCAGCTGCAAAGCCACCGGCGTAAGCGAGCCACGCCAGGCATCCAATACTGCAGCGATTCAGCAGAGAAGAACTGTTGCGTGCAGCCTCTTTATATTGACTTCCGTAAGGATTTGAACTGGAAATGGATCCACAAGCCCAGCGGCTACTATGCCAACTTCTGFβ2GGAGCTCTCCNNTATGGTCGACCTGCAGGCGGCCGCGAATTCACTAGTGATTATGCGCAAGAGGATCGAGGCCATCCGAGGGCAGATCCTAAGCAAACTGAACTCACCAGCCCTCCGGAAGAGTTCCCGGAGCCCGAGGAGGTTCCTCCGGAGGTCATTTCAATCTACAACAGCACAAGGGACCTGCTGCAGGAGAAAGCGAACCACAGGGCGGCCACTTGCGAAAGGGAGCGGAGCGAAGAGGAATATTACGCCAAGAGGTTTACAAAATTGATCTGCCTTTTTTCCATTCCGAAAACAAGATTCCACAAAAATCACCACAATCCAGCGCCTATTTTAAACGAGTGAGATTTGACGTCTCAGCAATGGAGAAAAATGCTTCCAACCTCGTGAAGGCAGAATTTAGAGTCTTTCGCTTGCAGAATCCAAAGGCTCGGGTATCAGAACAGCGGATAGAATTATATCAGATTCTCAAAAGCAAAGACCTAACTTCGCCAATGCAACGTTACATTGATAGCAAGGTAGTTAAAACACGA
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GCCGAAGGTGAATGGCTATCTTTTGATGTAACTGATGCTGTACATGAATGGCTCCATCATAGAGACAGGAATCTGGGATTTAAGATAAGTTTACATTGTCCATGCTGCACGTTCGTGCCTTCAAATAATTATATCATCCCAAACCAAAGTGAAGTGCTAGAAGCAAGTTTTGCAGGNATTGAGGACTACCACAGTGCTGAGAAAACTTTAAAGTCCAATNNNAAAAATACAGNGGGAAGACCCACATCTTCTGCTAATGTTGCTACCCTCCTACAGACTTGAGTCGCANCAGTCCAGTCNANGGANNANCGTGCTTTANATGCTGCCTNTGTTTAGNATGNGNANANACTGCTGNCTGCNACCATTTNCATTGATTCANANGGNNNNTGGNNGNAATGTGFβ3TTCGTACGCAGCAGTTCTTCTCCAGGTTTCGAAAGCAGTAATTGGTGTCTAGTGCTCGTTTTTTCCTTTGGCTTCTTACAGCTGGGCTCCCAAGCCTATGTGGGGGCAGCATCATCAAAATCAAGTGGGGATTGTGAAGGTCTTTCTGCTTCTTCAGGCTCCCTAAGTCCCCACGCCCATGGCTATCCTCGCTGTCGATGCCTTTGAACTTGATCTCCAGCACCTCATGCAGGTTCTCCAAGATGTCACCATTCGACTGGAAGGTATTACATGGGCAGTGGATGCTGATCTCCAGTCCCTGGTTAGATTCTCTATGCAAAAGCCATTCGCGCACGGTATCTGTGACATCAAAGGACAGCCACTCAGCGGAGCCCCGGGTCATCACGTTGCGGCCACTGAGGTAACGCTGCTTTGCAATATGCTCGTCGGGCTTAAGAATCTGGAAAAGCTCAATGCGTTGCTCGGTGCGTTTGGAACTGGGGTTTGGCACTCGCAAGACACGAAATTCAGCCCGGAAGAGGTTGGTGCTGTTCTTTTCTGCTGAAGACACGTTGAAGCGGAACACGTTGGAAGTTACCCCTTTGGGGCAGAATGTCAACTCGTTGTGCTCCGGGAGGCCCTGGATCATGTCAAATTTGTGGATCTCCTTGGCGTAGTACTCGGACTCGGTATTGTCCTGGGAGCAGCTCTCCTCCTTCTCGTCCTCCATCTCCTCCAGAAGCTCCCGGGTGCTGTTGTAAAACGCAATC18sACCCGGGGAGGTAGTGACGAAAAATAACAATACAGGACTCTTTCGAGGCCCTGTAATTGGAATGAGTACACTTTAAATCCTTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAA