A histological atlas of the tissues and organs of neotenic and metamorphosed axolotl

(1)

ActaHistochemica118(2016)746–759

ContentslistsavailableatScienceDirect

Acta

Histochemica

j ou rn a l h o m e p a g e :w w w . e l s e v i e r . d e / a c t h i s

A

histological

atlas

of

the

tissues

and

organs

of

neotenic

and

metamorphosed

axolotl

Turan

Demircan

(PhD)

a,d,∗

_,

_Ays¸

_e

_{Elif ˙Ilhan}

d

_,

_Nilüfer

_Aytürk

b,d

_,

_Berna

_Yıldırım

d

_,

Gürkan

Öztürk

c,d

_{, ˙Ilknur}

_Keskin

_(PhD)

_(MD)

b,d,∗

a_Department_of_Medical_Biology,_{International}_School_of_{Medicine, ˙Istanbul}_Medipol_University,_Istanbul,_Turkey b_Department_of_Histology_and_Embryology,_School_of_Medicine,_Istanbul_Medipol_University,_Istanbul,_Turkey c_Department_of_Physiology,_{International}_School_of_{Medicine, ˙Istanbul}_Medipol_University,_Istanbul,_Turkey d_Regenerative_and_Restorative_Medicine_Research_Center,_REMER,_Istanbul_Medipol_University,_Istanbul,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19April2016

Receivedinrevisedform7June2016 Accepted11July2016 Keywords: Axolotl Neoteny Metamorphosis Histologicalmap Thyroidhormones

a

b

s

t

r

a

c

t

Axolotl(AmbystomaMexicanum)hasbeenemergingasapromisingmodelinstemcellandregeneration researchesduetoitsexceptionalregenerativecapacity.Althoughitrepresentslifelonglastingneoteny, inductiontometamorphosiswiththyroidhormones(THs)treatmentadvancestheutilizationofAxolotl invariousstudies.Ithasbeenreportedthatamphibiansundergoanatomicalandhistological remod-elingduringmetamorphosisandthistransformationiscrucialforadaptationtoterrestrialconditions. However,thereisnocomprehensivehistologicalinvestigationregardingthemorphologicalalterations ofAxolotlorgansandtissuesthroughoutthemetamorphosis.Here,werevealthehistologicaldifferences orresemblancesbetweentheneotenicandmetamorphicaxolotltissues.Inordertoexaminestructural featuresandcellularorganizationofAxolotlorgans,weperformedHematoxylin&Eosin,Luxol-Fastblue, Masson’strichrome,Alcianblue,OrceinandWeigart’sstaining.Stainedsamplesfrombrain,gallbladder, heart,intestine,liver,lung,muscle,skin,spleen,stomach,tail,tongueandvesselwereanalyzedunderthe lightmicroscope.Ourﬁndingscontributetothevalidationofthelinkbetweennewlyacquiredfunctions andstructuralchangesoftissuesandorgansasobservedintail,skin,gallbladderandspleen.Webelieve thatthisdescriptiveworkprovidesnewinsightsforabetterhistologicalunderstandingofbothneotenic andmetamorphicAxolotltissues.

1. Introduction

Metamorphosistermis usedtodeﬁnetheinnate process of

amphibiantransitionfromlarvalstagetoadultform(Shi,2000).

Thistransformationprovidesanexcellentmodelsystemto

under-stand vertebrate organogenesis and remodeling of the organs.

During and following this transformation, commonly observed

phenotypicalchangesareanatomicalandhistological

reconstitu-tionoftheorgansaswellasappendagestofunctionproperlyin

terrestriallifeconditions.Regression,disappearingand/or

remod-elingoftheexistingorgansaswellasformationofneworgansare

theobservedadjustmentsofmetamorphosis(reviewedin(Brown

andCai,2007)).Forthedescriptionofchangesatorganand

sys-∗ Correspondingauthorsat:RegenerativeandRestorativeMedicineResearch Cen-ter,REMER,IstanbulMedipolUniversity,Istanbul,Turkey.

E-mailaddresses:tdemircan@medipol.edu.tr(T.Demircan), ilknurkeskin@medipol.edu.tr(˙I.Keskin).

temlevel,Xenopusleavisisthewidelyusedorganismamongthe

amphibians(BurggrenandWarburton,2007;Colomboetal.,2015).

Previous studieshave demonstrated thatfrom tadpoleto adult

frogtransformation,mostoftheorgansundergoremodelingsuch

asskin(Yoshizato,1996),lung(DoddandDodd,1976)andliver (Atkinsonetal.,1998).Theexternalgillsofthetadpoles,whichare

theprimarysiteforrespirationinaquaticenvironment,disappear

attheendofthemetamorphosis(Ishizuya-Okaetal.,2010).Bone

marrow,functionallimbsandglandsinskinandstomacharethe

examplesofnewlyformedcells,tissuesandstructureswith

meta-morphosis.Timingandrateofthiscomplexprocessisregulatedby

hormonalactivityandseveralexternalfactorssuchastemperature

(Hayesetal.,1993),densityofpopulation(SemlitschandCaldwell,

1982),threatofpredatorpresenceandfoodlevels(Kupferbergetal.,

1994).Intermsofhormonalregulation,thisremodelingcascade

startswithproductionandsecretionofthyroidhormones(THs).It

hasbeenfoundthat,THslevelsinamphibiansarelowatearlylarval

stageandpeakatmetamorphicclimax(MondouandKaltenbach,

1979).

(2)

SecretionofTHstobloodispursuedbyuptakingintothecells.Once

THlocateswithinacell,itbindstoitsreceptorscalledasthyroid

hormonereceptors(TRs)whichareasubclassofnuclear

recep-torfamilyproteins(Huangetal.,2010).Inmostofthevertebrates

therearetwoparalogousofthisgene;TRaandTRb(Escrivaetal.,

2002;ParisandLaudet,2008).IntheabsenceofTH,thesereceptors

aresuppressedbycorepressorproteinsandthereforetargetgenes

cannotbetranscribed.WhereasbindingofTHtoTRsbringsabout

theconformationalchangeoftheTRs,andreleasingofcorepressor

enhancesbindingofTRstohormoneresponseelements(HREs)on

DNAbyinteractingwithretinoidXreceptor(RXR)(Klieweretal.,

1992).BindingtoDNAtriggerstherecruitmentofcoactivator

pro-teins,andhence,expressionofthetargetgenesinthepresence

ofTHisachieved(Buchholzetal.,2006).Expressionofthegenes

withTHisessentialforremodelingoftheorgansduring

metamor-phosisofamphibians,andaccordingtomicroarraystudiesalarge

numberofgenesaredifferentiallyexpressedwiththeincreasedTH

activity(Dasetal.,2006;Yenetal.,2003).AlthoughTRsandRXR

proteinsarehighlyconservedamongthevertebrates,THinduced

geneexpressionproﬁleremarkablydiffersbetweentheanimals

(Bertrand etal.,2004).Inspiteofpresenceofconserved

coacti-vator,corepressorandnuclearreceptorsbetweentheamphibians

andmammals(FurlowandNeff,2006)limitedoverlapin

physio-logicalresponsetoTHbetweentheseanimalsindicatesspecialized

functionofTHinamphibians.

Unlikethefrogs, Axolotlrepresentslarvalcharacters beyond

thelarvalstage,throughoutitslife.InadequateconversionofT4to

T3,presenceofinactiveformofT3(3,3,5-triiodothyronine)and

expressionoflimitednumberofTRscontributeforlifelong

last-ingneotenyofAxolotl(Galton,1992).AdministrationofT4orT3

eitherbyinjectionorimmersionissufﬁcienttotriggerthe

meta-morphosis(Jacobsetal.,1988;PageandVoss,2009).Weightloss,

diminishmentanddisappearanceoftailﬁnandgillsandmolting

arethemorphologicalsignsformetamorphosis(Rosenkildeand

Ussing,1996).Availabilityofinductiontometamorphosisoffers

theopportunitytoutilizethemetamorphosedAxolotlasa

com-plementarysystemtoNeotenicones,sinceitisanaccomplished

modelorganismtostudyregeneration(CootsandSeifert,2015;

McCuskerandGardiner,2011;Vincentetal.,2015),scarlesswound

healing(Denisetal., 2013;Seifertetal., 2012), cancer(Menger

et al.,2010; Smithet al., 2000) and stemcells (RodrigoAlbors etal.,2015;Zielinsetal.,2016).Particularly,remarkable

regen-erationcapacityofthismodelholdsagreatpromisetounderstand

themolecularbasisofregenerationandrestorationconsideringits

successinfunctionalregenerationoftheinternalorgans(

Cosden-Deckeretal.,2012),centralnervoussystem(Amamotoetal.,2016; Madenetal.,2013;Zammitetal.,1993)andextremities(Kragletal., 2009;Satohetal.,2015)followingthedamageoramputation.

Con-sideringtheevolutionaryproximitybetweentheamphibiansand

mammals,employmentofAxolotlasamodelsystemallows

trans-lationofacquiredmessagestoMammalianseffectively.Although

numberofresearchesonAxolotlhasbeenexpanded,toourbest

knowledge,thereisnoextensivestudytogenerateahistological

mapofitsorgans.Here,inthisstudyweprovideahistologicalatlas

ofAxolotltissuesandorgansforbothpreandpost-metamorphic

stages.Allisolatedtissuesandorganswerehistologicallyanalyzed

useinfurtherstudies.Itiswellknownthathistological

documen-tationoftissuesandorgansistremendouslyusefultofollowupthe

effectsofanytreatmentsattissueandorganlevel.Therefore,we

certainlybelievethatthisreferencemapwillbeverybeneﬁcialand

bewidelyusedinAxolotlresearches.

2. Materialsmethods

2.1. Ethicalstatement

Animalcareandexperimentalprocedureswereapprovedbythe

AnimalResearchEthicsCommitteeoftheIstanbulMedipol

Uni-versity(authorizationnumber38828770-E.2302)andtheresearch

was performed in accordance with the European Community

guidelinesforethicalanimalcareanduseoflaboratoryanimals.

2.2. Animalhandlingandinductionofmetamorphosis

Axolotls (Ambystoma mexicanum) were obtained from the

AmbystomaGeneticStockCenter(AGSC)andbredinanimalcare

facilityof IstanbulMedipol University.Adult animals,14–16cm

in length, were used in all experiments. Animals were

main-tained in individual aquarias at ∼20◦_C _in _{Holtfreter’s} _solution

beforesampling.Metamorphosiswasinducedbyusing_l-thyroxine

(Sigma-Aldrich, T2376) as described below: (Page and Voss,

2009). T4 solution with a ﬁnal concentration of 50nM was

prepared by mixing l-thyroxine stock solution with modiﬁed

Holtfreter’s solution. Axolotls were transferred into containers

(oneAxolotl/container)having50nMT4solution.T4containing

mediumwaschangedeverythirddayandanimalswereobserved

formorphological changes.After∼2–3weeks ofT4

administra-tion,weightloss,disappearanceoftheﬁnanddecreaseinthegills

sizewereapparent.Administrationofthehormonewas

contin-uedforanother3weeksuntilfullymetamorphosedAxolotlswere

obtained.BothneotenicandmetamorphicAxolotlsweresacriﬁced

in0.02%benzocaine(Sigma-Aldrich,E1501)andorganswere

iso-latedimmediatelyafterthesacriﬁce.

2.3. Histologicalanalysis

Isolatedorgans(brain,gallbladder,heart,intestine,liver,lung,

spleen,stomachandtongue),skinandtailwereﬁxedin10%neutral

bufferedformalin(NBF)for48h.Followingtheremovalofﬁxative

bywashingthesampleswithtapwaterfor1h,theorganswere

incubatedinascendingalcoholseries(70%,90%and100%ethanol)

for 1hat 60◦C. Incubation in 100% ethanol wasrepeated two

moretimes.Then,sampleswereincubatedintoluenefor30minat

roomtemperaturetwice.Asanextstep,sampleswereembedded

toparafﬁn.Microtome wasusedtosectiontheparafﬁn

embed-dedorgansin4␮mthicktissuesections.Then,thesectionswere

deparaﬁnizedbyincubationintoluene(30minat60◦),

descend-ingalcoholseries(100%,96%and70%;1minatRT)anddistilled

water(1minatRT).Parafﬁnsectionsofallorganswerestainedwith

HematoxylinandEosin(Bio-OpticaMayer’sHematoxylinandEosin

(3)

748 T.Demircanetal./ActaHistochemica118(2016)746–759

Fig.1.Axolotlskinhistology.

MicroscopicexaminationofneotenicAxolotl’sskin.

A(10X)andd(10X)(hematoxylinandeosinstaining,bar=100␮m)E:Epithelium,M:Musculus,CT:ConnectiveTissue bande(10x)(massontrichromestaining,bar=100␮m)blackarrow:collagenﬁbers.

c(20x)andf(40x)(weigertstaining,bar=100␮m)blacktriangle:elasticﬁbers,circlesand*:mucousgland,quadrangleframe:Leydigcells.

histologicalstructures.Speciﬁctissuesinorganswere

character-izedbyLuxolFastBlue(KIT,LuxolFastBlueKluverveBarrera,Bio

Optica,04-200812),Masson’sTrichrome(KIT,MassonTrichrome

withanilineblue,BioOptica,04-010802),AlcianBlue(KIT,Alcian

BlueAcidMucopolusaccharidesstaining,Bio-Optica,04-160802),

Weigert(KIT,WEIGERT-VANGIESONforelasticﬁbersand

connec-tivum,BioOptica,04-053812)andOrcein(KIT,OrceinforElastic

Fibers, Bio Optica, 04-055802) byfollowing themanufacturer’s

suggestedprotocol.Bio-mountsolutionwasusedtocovertheall

stainedsampleswithcover-slide.Theimagingwasperformedby

usingtheNIKON DS-Fi2-U3 DigitalCameraand ImageAnalysis

SoftwareSystem.

3. Results

Theprocessofamphibianmetamorphosisischaracterizedby

plenteousmorphological and biochemicaltransitions which are

mainlyresponsibleforadaptingthenewterrestrialenvironment.

Inthisdistinctivephaseoftheirlifemostorgansandextremities

suchasspleen,liver,skinandtailareremodeledsothattheaquatic

organismaccustomstobeaterrestrial.Tissuesandorgansare

clas-siﬁedintotwo groupsbasedonthemajororminorremodeling

events.

3.1. Majorremodelingevents

3.1.1. Skin

Skinsamples were obtainedfrom the backskin of the

ani-mals. Histological examination of the skin presents two main

layers;epidermisanddermis.Thicknessoftheselayersoftheskin

isdependenttoandanindicatorofregionalvariation.Neotenic

axolotlepidermisiscalledpseudo-stratiﬁedepitheliumand

con-tainsepithelialandLeydigcells(Fig.1a–c).Secretingglandsformed

byinvaginationofepidermallayerthroughdermis(Fig.S1a).The

dermisis constructedby irregularloose connectivetissue.

Fur-thermore,collagenﬁbersandatraceofﬁbroblastsareobserved

indermis(Fig.1b,c,S1b).

Noticeabledifferencesinskinorganizationofmetamorphosed

Axolotlareobserved(Fig.1d).Asasignofadaptationtoterrestrial

lifeconditions,theepidermisisformedofkeratinizedstratiﬁed

squamousepithelium(Fig.1d–f)liketheotherterrestrial

(4)

Fig.2. Axolotltailhistology.

Remodellingoftailduringmetamorphosis.MorphologicalalterationsbetweentheneotenicAxolotl’stail(a–c)andmetamorphicAxolotl’tail(d–f)areshownontaken sections.

a(4x,bar=1000␮m)andd(10X,bar=100␮m)(hematoxylinandeosinstaining)E:Epithelium,M:Musculus,CT:ConnectiveTissue,blackcircleandSc:spinalcord, not:notochord,

*:mucousandgranularglands,pca:perichordalcartilage

b(4x,bar=1000␮m)ande(10x,bar=100␮m)(massontrichromestaining)E:Epithelium,M:Musculus,CT:ConnectiveTissue,blackcircle:spinalcord,not:notochord,*: mucousandgranularglands,

c(4x,bar=1000␮m)andf(10x,bar=100␮m)(Luxolfastbluestaining)E:Epithelium,M:Musculus,CT:ConnectiveTissue,blackcircleandSc:spinalcord,not:notochord, pca:perichordalcartilage,crd:chordoidcells/tissue.

epidermiswithoutskinappendages(hair,sebaceousglands,sweat

glands)isobserved.ItisnotedthatLeydigcellsdisappearedand

theepidermiscontainedawell-deﬁnedstratumspinosum,

gran-ulosumandcorneum(Fig.S1c,d).Dermalpapillaisnotobserved.

Mucousglandsarefoundathighnumbers(Fig.1d–f).Alignmentof

collagenﬁbersinthedermisisobservedmorecloselyandintensely

(Fig.S1d)thantheneotenicskinsample.Especiallytheallocation

ofelasticﬁbersisrecognizedaroundthemucus-producingglands

(Fig.1f).

3.1.2. Tail

NeotenicAxolotls’tailhaspseudo-stratiﬁedepitheliumwhich

includes epithelial and Leydig cells similar to skin epithelium

(Fig.2a).Beneaththeepithelium,inconnectivetissue,thereare

skeletalmusclesshapedassigniﬁcantfascicules(Fig.2a,b)

encom-passedbyakindofconnectivetissuecalledperimysium(Fig.2a,b).

Inthemiddleofthetailsection,notochordisnoticed(Fig.2a–c)and

thechordoidcells/tissueinthecenterofthenotochord[structures

aredescribedin(Jonassonetal.,2012;Schnappetal.,2005)].

Chon-drocytescanbedetectedincartilagetissue(Fig.2c).Moreover,the

spinalcordisalsomarkedintailsectionasexpected(Fig.2c).

Asaresultofmacroscopicalteration,ﬁnisdisappearedafter

metamorphosis.Basedonlightmicrocopyresults,themain

adap-tation in tail epithelium is conversion to keratinized stratiﬁed

squamous epithelium(Fig.2d, e).Presenceofmucous glandsis

recognizedunderneaththeepithelium(Fig.2e).Spinalcord,

noto-chord,andatthecenterofnotochordthechordoidcells/tissueare

observedinmetamorphicAxolotlstailsectionwhichresemblesthe

neotenictailsamplesection(Fig.2f).

3.1.3. Spleen

InneotenicAxolotlsspleenweobservedintensivecellregionsas

awhitepulp–probablyincludeslymphocytes–whichareplaced

aroundthecentralvein(Fig.3b)asdescribed elsewhere(Lopez

etal.,2014).Redpulpsarealsonoticedwhichconsistsofredblood

(5)

Fig.3.Axolotlspleenhistology.

Microscopyofneotenic(aandb),andmetamorphicAxolotl’sspleen(candd).

Histologicalsliceswerestainedwithhematoxylinandeosinandpresenceofbloodcells(a)redandwhitepulpsinneotenicspleenwasnoticed(b). a(10x,bar=100␮m)andc(4X,bar=1000␮m)(hematoxylinandeosinstaining)C:Cortex,blackarrow:bloodcells,T:Trabecula.

b(20x)andd(20x)(hematoxylinandeosinstaining,bar=100␮m)blackarrow:bloodcells,RP:Redpulp,WP:Whitepulp.(Forinterpretationofthereferencestocolourin thisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

coulddetectbloodcellsandtrabeculastructure(Fig.3c,d).

How-ever,we couldnot signiﬁcantly recognize white and redpulps

(Fig.3d).

3.1.4. Gallbladder

Gallbladder hasseverallayers;themucosa, muscularis,

per-imuscularandserosa.Our resultsuggeststhat therearecertain

structuraldifferencesbetweenneotenicandmetamorphicAxolotls

gallbladder.

Firstof all,limitedmucosalfoldsareobserved inthelumen

oftheneotenicAxolotlsgallbladder(Fig.4a)whereasnumberof

mucosalfoldsincreasesaftermetamorphosis(Fig.4b).Although

epitheliumandlooseconnectivetissueexist,thereisnomuscle

layerbeneath theepithelium for neotenicgallbladder (Fig. 4a).

Ontheotherhandinmetamorphosedgallbladdercircularsmooth

musclelayerdispersedinvesselrichconnectivetissueisobserved

(Fig.4b,c).Moreover,NeotenicAxolotl’sepitheliumis

character-izedbysingle-layeredcubic/single-layeredprismaticcellsandafter

metamorphosisgallbladderepitheliumofAxolotlchangeto

multi-layeredfromsingle-layered(Fig.4b).Furthermore,unlikeneotenic

Axolotl,Rokitansky-Aschoffsinusesformedbymucosalfoldscan

beseeninmetamorphicorganism(Fig.4b).

3.2. Minorremodelingevents

3.2.1. Cerebrum

Wecouldnotdetectdramaticdifferencesbetweentheneotenic

andmetamorphicAxolotlsbrainsections.Theaxolotlsbrainis

char-acterizedbythepresenceofanarrow,one-tothreecelllayeredVZ

(matrixzone)contiguoustotheventricle(Fig.5b).TheVZis

encom-passedbywide regionof uniformly sphericalneurons((Maden

etal.,2013);Fig.5b).AsshowninFig.5,Granulecelllayer(GcL),

MitralcellLayer(McL)andGlomerularLayerexistinbothneotenic

andmetamorphicAxolotlsbrainsections(a–d).Theolfactorybulb

(Fig.5a)andAnteriorOlfactoryNucleus(Fig.5b)arenoticedon

neotenicAxolotlsbrainsection.

3.2.2. Tongue

Asshown intheﬁgures nosigniﬁcantstructuraldiversity is

observedbetweentheneotenicandmetamorphicAxolotltongue

sections(Fig.6a–d).Inbothofthesamples,anon-stratiﬁed

squa-mousepitheliumcoversthelooseconnectivetissue (Fig.6a–d).

Likewisetoskinepithelium,Leydigcellsexistwithinthe

epithe-liumoftongue(Fig.6b,c).Bothtonguesectionshaveavesselrich

connectivetissue(Fig.6b,c)whichisalsositefortheskeletalmuscle

ﬁbers(Fig.6c,d).Furthermore,thehyalincartilageareasarenotably

observedinbothneotenicandmetamorphicAxolotlstongue

sec-tions(Fig.6a,d).

3.2.3. Heart

Asshown in Fig.7 there is nodramatic difference between

neotenicandpostmetamorphicAxolotlsheartsectionsinterms

ofcardiomyocytedispersionandorganizationofthetissue.

Car-diomyocytescanbeobservedinrandompatterninbothanimals

(Fig.7a,c).Thesecellshaveacentralovalnucleusandtheyform

thestriated heartmuscle structure (Fig. 7b,d).As distinctfrom

postmetamorphic animal’sheart section, theconnective tissue

(endomysium),surroundedbymuscleﬁbers,ismorenoteworthy

inneotenicone(Fig.7b,d).

3.2.4. Lung

Accordingto our result, central air space of Axolotl lung is

dividedintosmallerairpocketsbyalveolarfolds(Fig.8a–c).Blood

vessels,alveolarfoldsandsmoothmuscletissuesformanetwork

tofacilitatethegasexchange.Thinepitheliumofthealveolarfolds

(6)

Fig.4. Axolotlgallbladderhistology.

Neotenic(a)andmetamorphicAxolotl’sgallbladder(bandc).Epithelialandmuscletissuesareformed/reformedduringthemetamorphosisprocess(bandc). a(10X),andb(20X)(hematoxylinandeosinstaining,bar=100␮m).

thickblackarrow:epithelium,CT:connectivetissue,thinblackarrow:smoothmuscle, *:Rokitansky–Aschoffsinuses.

c(20X)(massontrichromestaining,bar=100␮m),v:vessel.

Fig.5. Axolotlcerebrumhistology.

Microscopicexaminationofneotenic(aandb)andmetamorphicAxolotl’scerebrum(candd).

a(10X),c(10X)andd(20X)(hematoxylinandeosinstaining,bar=100␮m)ob:olfactorybulb,GcL:Granulecelllayer,McL:Mitralcelllayer,GL:Glomerularlayer,AON:Anterior olfactorynucleus.

b(10X)(luxolbluestaining,bar=100␮m)v:ventricle.

threedifferentcelltypeswhicharepneumocytes,ciliatedcellsand

gobletcells(DierichsandDosche,1982).WhilePneumocytesare

locatedononesideofcapillary(Fig.8b),thetwoothergroupsof

thecells,ciliatedandgobletcells,coverthesmoothmusclecells

anddonottakepartintherespirationprocessdirectly(Fig.8a,c).

Bloodcellsaredetectedinthelargebloodvesselsandvoluminous

connectivetissueisrecognized(Fig.8b,c).

Weobservedsomesimilaritiesandvariationsinneotenicand

metamorphicAxolotl’slungsamples.Themajornoticeddifference

isanobviousdecreaseintheamountofconnectiveandsmooth

muscletissues(Fig.8d,f).Furthermore,althoughthepneumocytes

andciliatedcellsarerecognizedaroundtheairpockets(Fig.8e),we

couldnotdetectanygobletcells(Fig.8f)onthesection.Thereare

alsomanyalveoliwhichhasafunctioninpulmonaryrespirationin

metamorphicAxolotlasinmammalianlungs(Fig.8f).

3.2.5. Liver

Themicroscopicimagesofhistologicalslidesofneotenicand

metamorphicAxolotls’liveraredepictedinFig.9.Asshowninthe

ﬁgure,therearenovoluminousdifferencesbetweentheliverof

neotenicandmetamorphicorganismintermsoftissuecomposition

andcelltypes.Theobserveddifferencescanbelistedasfollowing:

Firstofall,neotenichepatocytes(shownwith‘h’letteronﬁgures)

arepolyhedralandhavearoundednucleus(Fig.9a,b),whilethe

hepatocytesofmetamorphic organismsareseenaseither

poly-hedralorrounded(Fig.9c,d).Furthermore,neotenichepatocytes’

nucleiarelocatedinthecenterofcytoplasmorapproachtoone

sideofcytoplasm.Ontheotherhand,aftermetamorphosis

hepa-tocytesbecomemoreregularandnucleiofthecellsarecommonly

locatedinthemiddleofcytoplasm.Themostsigniﬁcanthistological

(7)

Fig.6.Axolotltonguehistology.

Microscopyofneotenic(aandb)andmetamorphicAxolotlstongue(candd).

a(4x,bar=1000␮m)andc(20X,bar=100␮m)(hematoxylinandeosinstaining)blacktriangleEpithelium,*:Cartilage,CT:ConnectiveTissue,M:Musculus,blackarrow: Leydigcell.

b(40x)andd(10x)(massontrichromestaining,bar=100␮m)blacktriangle:Epithelium,*:Cartilage,CT:ConnectiveTissue,M:Musculus,blackarrow:Leydigcell,V:Vessel, thinarrow:denseregularconnectivetissue.

Fig.7.Axolotlcardiactissuehistology.

Morphologicalanalysesofneotenic(aandb)andmetamorphicAxolotl’scardiactissue(candd). a(10x)andb(10X)(hematoxylinandeosinstaining,bar=100␮m).

c(40x)andd(40x)(hematoxylinandeosinstaining,bar=100␮m)blackarrowindicatesthecentralnucleusofcardiacmusclecells.

incytoplasmicstaining.Inneoteniclivertissueabsenceofregular

stainedareasispresumablyduetocytoplasmicglycogenandlipid

storage.Additionally,alargenumberofmelaningranules

assem-bliescanbenoticedinthemetamorphicAxolotl’sliverparenchyma,

asopposedtoneotenic(Fig.9c).Centralveinofneotenicand

meta-morphosedliversamplesresembleeachotherand elasticﬁbers

arenoticedaroundthecentralveininbothtissues(Fig.9b,d).Our

(8)

Fig.8. Axolotllunghistology.

Neotenic(a,bandc)andmetamorphicAxolotl’slungsections(d–f)indicatesstructuralsimilaritiesandvariationsbeforeandafterthemetamorphosis.

a(10X)andd(10X)(hematoxylinandeosinstaining,bar=100␮m)V:Vessel,ap:airpocket,M:smoothmussle,a:alveol,blackarrow:ciliatedandcuboidalcells,CT:connective tissue.

b(20X)ande(20X)(massontrichromestaining,bar=100␮m)V:Vessel,ap:airpocket,a:alveol,blackarrow:ciliatedandcuboidalcells,blacktriangle:pneumocytesM: musculus.

c(20x)andf(20X)(alcianbluestaining,bar=100␮m)V:Vessel,thinblackarrow:gobletcell,ap:airpocket,a:alveol.(Forinterpretationofthereferencestocolourinthis ﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

thepreviouslyobservedandreportedimagesand/ordescription

(Lopezetal.,2014).

3.3. Stomach

Thetypicalhistologyof stomachis comprisedoffourlayers.

AsdemonstratedinFig.10,fromtheinsideout,theselayersare

mucosa,submucosa,muscularlayerandserosallayer;respectively.

Themucosaisformedofmucous-secretingcolumnarepithelium

andnumerousgastriccells(Fig.10a,d).Gastricchiefcellsareone

ofthemaincomponentsofmucosalayerforbothneotenicand

metamorphicanimalstomach(Fig.10b,e)Betweenthemucosaand

epitheliuminlaminapropria,serousandmucousglandsarenoticed

(Fig.10b,e).Mucin-secretinggobletcellsarefoundin

metamor-phicAxolotlstomachsample(Fig.10f).Connectivetissuegetsmore

organizedaftermetamorphosisasshowninsubmucosa(Fig.10e)

Incontrary,fortheneotenicones,connectivetissueelements

dis-persedmorebetweenthegastriccells thanthemetamorphosed

animals(Fig.10b, e) Submucosa,characterizedby vascular and

undifferentiatedconnectivetissues,isdetectedinboth neotenic

andmetamorphic Axolotl(Fig.10a–f).Intensevascularizationis

noticedinsubmucosainbothorganisms(Fig.10c,f).Finally,thelast

observationaboutbothneotenicandmetamorphicAxolotl

stom-achis,tunicamuscularis(shownintheﬁgureasME)composedof

circularsheet.

3.4. Intestine

AsshowninFig.11,luminalsideoftheintestineiscomposed

ofmultilayerofepithelialcells.Theepitheliumissurroundedby

thinlayersofconnectivetissueandoutermuscles.Thereis

numer-ousepithelialfold.Theneotenicintestinaltractresemblesatypical

vertebrateintestine.Theepitheliumisintoathicktemporarymulti

cellularlining(Fig.11a–c).Theepitheliumhasabundantgobletcells

(Fig.11b,c).

Intestine of metamorphosed animal shares similarities with

neotenicones.Forinstance,theepitheliumoftheintestineis

devel-opedintothemultiplyfoldedstructure(Fig.11d).Cryptandvilli

(9)

plen-754 T.Demircanetal./ActaHistochemica118(2016)746–759

Fig.9.Axolotlliverhistology.

Microscopicexaminationofneotenic(aandb)andmetamorphicAxolotl’sliver(candd)ontakensections.

a(20x)andc(20X)(hematoxylinandeosinstaining,bar=100␮m)h:hepatocytes,CV:centralvein,blacktriangle:sinusoid,C:Cortex,blackcircle:Brownpigmentgranules. b(10x)andd(20x)(massontrichromestaining,bar=100␮m)blackarrow:basementmembrane,h:hepatocytes,CV:centralvein,blacktriangle:sinusoid.(Forinterpretation ofthereferencestocolourinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

tifulgobletcellsaredetectedinmetamorphosedsamplesaswell

(Fig.11e,f).

Isolatedintestine sizeforthemetamorphosed oneis

signiﬁ-cantlyshorterthantheneotenicone(datanotshown).

3.5. Skeletalmuscle,vesselandbonetissues

Axolotlsskeletalmusclesectionsareindistinguishablefromthe

mammalianskeletalmusclesintermsofthenucleuslocalization.

Asshownintheﬁgureoftransversalandlongitudinalmuscle

sec-tions,thenucleusisplacedtheperipheryofcell(Fig.S2a,c).Muscle

fasciclesaresurroundedbyconnectivetissue(Fig.S2b,d)and

meta-morphicmusclefasciclesaremorecompactthantheneotenicones

(Fig.S2b,d)asdeﬁnedelsewhere(Monaghanetal.,2014).On

meta-morphicsection,therearemoreintensiveconnectivetissuethan

inneotenicone,andasastrikingdifference,lossofedemainthe

connectivetissueisobserved(Fig.S2b,d).

Beside analyzedmuscle tissue, nosigniﬁcant alterations are

noticedforvessel(Fig.S3) andbone (Fig.S4) sections.For

ves-selsamples,artery,vein,erythrocytes,endotheliumandﬁbroblast

cellsaredetectedinbothneotenicandmetamorphicAxolotls(Fig.

S3a–d).Inthesameway,wecouldnotrecognizecrucialdifferences

inbonesamples.Vessel,osteocytes,collagenandcartilagetissue

arefoundinbothneotenicandmetamorphicbonesections(Fig.

S4a,b).

4. Discussion

4.1. Skin

Skinistheprotectivecoverageforthebodysurfaceandit

con-ductsmanyvitalfunctions.Foraquaticorganisms,skinpermitsthe

transitionofoxygenandwaterbetweentheorganismand

envi-ronmenttomaintainthehomeostasis.InneotenicAxolotl,oxygen

transportthroughtheskincontributes torespirationand

pene-trationofwaterintotheskinfulﬁllstheneedtowater.Themost

strikingchangeswiththeinductionofAxolotltometamorphosis

occurintheskin.Inpreviousstudiesremovalofpre-metamorph

epidermisandconstructionofmetamorphepidermisasa

conse-quenceofTHsinductionwasexamined(Pageetal.,2009;Seifert

etal.,2012)andtheirresultsareagreeswellwithour

observa-tions.StructureofmetamorphosedAxolotlskinresemblesother

terrestrialvertebrates’keratinizedstratiﬁedsquamousepithelium.

Thisadaptationiscrucialtoreducethewaterlossandprotectthe

organismfromphysicaldamage.Humidityleveloftheskinis

main-tainedbycontinuousmucussecretionfromtheincreasednumber

of glands. Moreover,this increase mayaccount for providing a

protectivelayer.Additionalrolesthatmucussecretionplaysare

protectionfrompathogenssuchasbacteria,coolingthebodyvia

evaporationandfunctioningasapartofexcretionsystem.

4.2. Tail

ChangingsbetweenneotenicandmetamorphicAxolotlstail

sec-tionsaresimilartoalterationsintheskin.Theepitheliumturnsinto

keratinizedstratiﬁedsquamousepitheliumandmucousglandsare

formedtoactinrequiredmucoussecretionfortheterrestriallife

conditions.Sinceaquaticanimalsdonotneedtohumidify their

skin,appearanceofmucousglandsaftermetamorphosisis

essen-tialtopreventwaterloss.Besidethehumidityrelatedremodeling

andlossoffin,nosignificantmodificationsarenoticed.

4.3. Spleen

Thespleenisoneofthelymphoidsystemorgansanduntilﬁfth

monthoffetallifeitactsasapartofhematopoieticsystem.On

post-partumperiodinvertebrates,spleenhasvarioustasksintermsof

bloodﬁltrationandstorage,phagocytosis,destructionofold

(10)

Fig.10.Axolotlstomachhistology.

Microscopyofneotenic(a,bandc)andmetamorphicAxolotl’sstomach(d–f)ontakensections.

a(10X)andd(10X)(hematoxylinandeosinstaining,bar=100␮m)M:Mucosa,GC:Gastriccells,SM:Submucosa,ME:Muscularisexterna.

b(20X)ande(20X)(massontrichromestaining,bar=100␮m)E:Epithelium,blackarrowsshowparietalcellsandblacktriangleindicateschiefcells.

c(20X)andf(20X)(alcianbluestaining,bar=100␮m)circle:gobletcells,*vessel.(Forinterpretationofthereferencestocolourinthisﬁgurelegend,thereaderisreferred tothewebversionofthisarticle.)

The spleen is surrounded by a capsule and from this

cap-sule,thinconnectivetissuecompartments(trabecula)splitoffand

thesecompartments proceedtodepthof theorgan withblood

cells.Capsuleandtrabeculaeformthestromaofthespleen.The

spleenparenchymasettledinthisstromaiscalledthepulp.The

pulp is shaped by cell rich connective tissue and divided into

two groups; white and red pulp. White pulp consists of cords

formedbylymphoidtissue.Redpulpisaspeciallymphoidtissue

includesreticulumcells,macrophages,plasmacells,lymphocytes,

andplatelets(EroschenkoandDiFiore,2013)

InneotenicAxolotlsspleen,whitepulpanderythrocytesrich

red pulp areas indicate that spleen takes an active role in

hematopoiesis.Aftermetamorphosisnotwithstanding,we

consid-eredthespleenmayalreadyhavecompletedthisfunction.

4.4. Gallbladder

Weobservedmajordifferencesindifferentlayersofthe

gall-bladderbeforeandafterthemetamorphosis.Themucosalayerof

gallbladderconsistsofasimplecolumnarepitheliumandunderline

theepitheliumthereisconnectivetissue.Nosubmucosaisdeﬁned

ingallbladder.Changeofepitheliumorganizationandincreasein

themucosafoldsmightpointoutthefactthatbileisconcentrated

ingallbladderafterproductionintheliverforthemetamorphosed

animalbutnotfortheneotenicone.Themuscularislayeris

com-posedofscatteredbundlesofsmoothmuscle(Rajguruetal.,2013).

Adventitalocatesin themuscularisand denseconnectivetissue

ofAdventita bindsthegallbladder totheliver.Gallbladder

sur-faceandabdominalcavityisinterruptedwithserosalayerwhich

comprisesbloodvessels,nervesandalymphaticnetwork(Frierson,

1989).Theexistenceofmusclelayeraftermetamorphosiscanbe

interpretedasacrucialalterationinordertoconcentratebile

secre-tion.LackofmusclelayeringallbladderofneotenicAxolotlcould

beduetotheunnecessityofactivebilesecretioninthisperiod.

Rokitansky-Aschoffsinusesarepseudo-diverticulainthewallof

gallbladderandformationofthemmightberelatedtoincreased

pressure(Rajguruetal.,2013).

4.5. Cerebrum

Inmammals,THsarecrucialfor braindevelopment.

Regard-ingthepreandpostmetamorphicstages,amphibiansrepresentan

excellentmodeltoinvestigatetheTHsrolesonbraindevelopment

(11)

develop-756 T.Demircanetal./ActaHistochemica118(2016)746–759

Fig.11.Axolotlintestinehistology.

Neotenic(a–c)andmetamorphicAxolotl’sintestinesections(d–f)speciﬁesstructuralresemblancesanddifferencesbeforeandafterthemetamorphosis. a(20X)andd(20X)(hematoxylinandeosinstaining,bar=100␮m)S:Serosa,SM:Submucosa,M:Mucosa,V:Vessel,L:Lumen,blackframe:villi. b(20X)ande(20X)(massontrichromestaining,bar=100␮m)SM:Submucosa,M:Mucosa,L:Lumen,blackarrow:gobletcells,*:musculus. c(20x)andf(20X)(alcianbluestaining,bar=100␮m)S:Serosa,M:Mucosa,V:Vessel,L:Lumen,blackarrow:gobletcells,*:musculus.

ment.Additionally,amphibiansVentricularzone(VZ)andNeural

progenitor cells (NPCs) are considered to proliferate

through-outadulthoodwhichisnotcommonforhighervertebrates.The

amphibiantelencephalon includesa dorsal and thicker ventral

matrix (ventricular) zone that reveals higher proliferative and

regenerativecapacitythantheteleostandreptiletelencephalonVZ

(Madenetal.,2013).Incomparisontoanuranmodels,an

impor-tantbeneﬁtinemployingAxolotlistheabilitytoinducetheonset

ofmetamorphosisinjuvenileoradults(Hugginsetal.,2012).From

thesectionsweexamined,wedidnotobserveanydrasticchanges

betweenneotenicand metamorphic brainslices.This mightbe

theresultofearlymodelingofbrainwithlimitedTHspresentin

neotenicAxolotl. Inorder totest this hypothesis,it isworthto

analyzebrainofyoungerjuvenileAxolotls.Moreover,histological

resemblancesofmetamorphosedbrainareencouragingtoinspect

moredetailed for regenerationcapacity asdescribed elsewhere

(Amamotoetal.,2016).

4.6. Tongue

As seen in some other organs, there is no detectable

dif-ferencebetweentheneotenicand metamorphicAxolotl tongue

samplesbasedonhistologicalstainingandlightmicroscopyresults.

Absence of lingual papillae in both samples is a noteworthy

observationincomparison tomammaliantonguestructure.The

mammaliantongueisorganizedascoreofmusclecoveredby-non

stratiﬁed/stratiﬁedsquamousepithelium.Thisepitheliumlayeris

coveredwithlingualpapillaecharacterizedbyvarious

irregulari-tiesandelevations.(BillinghamandSilvers,1967).LackofLeydig

cellsinlarvalAxolotlandappearanceduringthedevelopmentmay

correlatewithpartialreorganizationoftissuesviasecretionofTHs

(Wistubaetal.,1999).WedetectedLeydigcellsinbothneotenic

andmetamorphosedAxolotl’stonguesections.Remarkableﬁnding

aboutthemetamorphosedAxolotltongueispresenceoftheLeydig

cellssincetheonesintheskindisappearduringmetamorphosis.

4.7. Heart

We did not observe considerable variations between the

neotenic and metamorphic heart sectionsregarding

cardiomy-ocytes. However,Malvinand Heisler pointed outoccurrence of

severalmorphologicaldifferences of heartafter metamorphosis

(MalvinandHeisler,1988).Themajoralterationthatthey

(12)

approximately%45oftotalO2uptakeactualizeswithpulmonary

respirationinneotenicstage,aftermetamorphosisthisproportion

increasessigniﬁcantly andbecomes approximately%65 (Malvin

andHeisler,1988).Presumably,theobservedsimilaritybetween

theneotenicand metamorphic Axolotlslung sectionsis dueto

activeusageoflungsduringtheneotenicstage.Thisisanotherlayer

ofevidencetosupportthepartialremodelingoftheneotenicorgans

withtheproducedT3hormonewhoselevelisnormallyinadequate

toinducemetamorphosis.

However, the transition from aquatic to terrestrial life still

obligesseveralcrucialadaptationsregardingrespiration.For

exam-ple, theloss of buoyancy requires more energy for movement

and consequently an increased metabolism with increasedgas

exchange.Since terrestrial animals’skinis adaptedtodiminish

thewater loss,this alteration bringsabout thedecrease ingas

exchangethroughtheskin.Lossofgillduringmetamorphosisis

anotherreasontoberestrictedtouselungsmoreefﬁcientlyforthe

metamorphosedanimals.Toovercomethedifﬁcultiescomingwith

terrestriallifeconditions,numberofalveolarincreasesandshows

morefoldingtoincreasethesurfaceareainordertocarryoutan

effectiverespiration.

4.9. Liver

Thelargestinternal organis liverand it hascrucial rolesin

proteinsynthesis,storageofmetabolites,bilesecretionand

detox-iﬁcation. Lobule structure of liver is formed from hepatocytes

andcapillarynetworkcalledassinusoidswhicharelocalizedin

betweenhepaticplates.Accordingtolightmicroscopyresults,we

didnotdetectremarkabledifferencesbetweenneotenicand

meta-morphosedAxolotlliversamples.Themaindistinctionnoticedis

incytoplasmicstainingpattern,whichcanbeduetoglycogenand

lipidstorageinneotenicIivertissuesandnotinmetamorphosed

ones.Variations in feedingregime with metamorphosisand/or

temperaturedifferenceinandoutofaquaticenvironmentmaybe

thesourceofobserveddifference.

Clusteringofmelaningranulesinmetamorphicliversamples

mayberelatedtohigherphagocyticactivitysincehepatocyteswith

melaninpigmentsareconsideredasthepartofreticulohistiocytic

system.Difﬁcultiesinremovalofcytotoxicionsandfreeradicals

fromthebodyinterrestriallifeconditionsmayaccountforhigher

phagocyticactivityandthereforeclusteringofmelaninpigments.

4.10. Stomach

Thestomachisplacedintheposteriorforegutandits

morphol-ogyidentiﬁedwiththickenedmuscleanduniqueglands.Thickened

muscleisessentialforperistalticmovementsandelasticdistention

whenthestomachisﬁlledwithlargequantityoffood(Smithetal.,

2000).

Accordingtopreviousresearches,mostanurantadpoles

gen-erallyindicateslackinga“true”stomachandproteolyticenzymes

(Smithetal.,2000).However,gastricchiefcellswhichproduce

pro-teolyticpepsinogenenzymecanbenumerouslyseeninneotenic

Axolotlstomach.Itcouldbetheresultofbeingacarnivoreborn

species,andthereforerequiredenzymestohydrolyzetheproteins

4.11. Intestine

Intestineisoneofthehighlyplasticorgansthatfunctionsmainly

infoodprocessingand nutrientabsorption.Itownsits

reestab-lishment to its plasticity and this is highly affected from THs.

Postembryonicdevelopmentandremodelingofintestineisa

com-monlyobservedprocessamongvertebrate.Inmouse,formation

ofcrypt-villistructure takesfew weeksafterthebirth sinceT3

production and its release to blood is required for remodeling

theintestine(Hasebeetal.,2013).T3isalsocrucialfor

amphib-ians’ intestine establishment post-embryonicallyand itsclimax

structures the crypt-villi formation in intestinal epithelium of

amphibians(SunandShi,2012).

Basedonourlightmicroscopyresults,thereisnosigniﬁcant

dif-ferencebetweentheneotenicandmetamorphicAxolotlintestine,

unlikethefrog.IthasbeendocumentedthatT3isproducedbythe

timeofearlyjuvenilestagesinneotenicAxolotlbutitisnotenough

tostimulatethemetamorphosis(Badawy,2011;Rosenkildeand

Ussing,1996).Sincetheanimalsweusedwereolderthan6months,

themostplausiblescenarioissecreted T3amountistoolow to

transformtheanimals;howeveritishighenoughtoremodelthe

intestine.Inthatrespect,post-embryonicintestinalestablishment

ofneotenicAxolotlshouldoccuratveryearlystagesafterthebirth

oftheanimal.

4.12. Skeletalmuscle,vesselandbonetissues

In both neotenic and metamorphic animals skeletal muscle

andconnectivetissuesexhibitsimilaritiesratherthandifferences

in terms of found cells and structural organization. The main

alterationinthesetissuesislossofedemawithTHsclimax.

Resem-blancesofthesetissuesbetweenpreandpostmetamorphicanimals

mightbeduetoconservedstructuralrolesofthesetissuesamong

vertebrates.

5. Concludingremarks

Tissue renewal and restoration capacity of Axolotls makes

thempromisingmodeltoexplorethemolecularmechanismsthat

havecrucialrolesinregeneration.Availabilityofmetamorphosed

AxolotlsbyadministrationofTHsandalterations in

experimen-talcharacteristicsaftermetamorphosisoffersadualmodelsystem

to performstem cell, regeneration and cancerstudies. Reliable

evolutionaryproximitybetweenamphibiansandmammalsmay

facilitatethedigestionofmessagesfromsalamanderstudiesandit

mayenhancethetranslationofmessagesfromamphibiansto

mam-mals.Inthisstudywepresentacomparison ofneotenicaxolotl

withmetamorphiconesintermsofdetailedhistologicalanalysis

whichmaycontributetogainnewperspectivestolinkthe

renew-ablecapacityoftissuesandtheirorganizationforthefuturestudies.

Ourﬁndingssuggestthatremodelingoforgansandtissueswith

THsinductionfacilitatetheadaptationtoterrestriallife.

Surpris-ingly,wehavenoticedextensivesimilaritiesbetweentheneotenic

(13)

inearlydevelopmentalstagesmayaccountforremodelingof

sev-eraltissuesandorgansofNeotenicAxolotlduetobeingresponsive

totraceamountsofTHs.Totestthispossibility,amore

compre-hensiveresearchbyinspectionoforgans fromyoungeranimals

atdifferentdevelopmentalstagescanbecarriedout.Moreover,a

moredetailedanalysisusingelectronmicroscopytoexamineintra

andintercellular componentswouldbeadvantageousto

distin-guishresemblingstructures.Furthermore,cellandtissuespeciﬁc

immune-stainingwouldprovideavaluabledatatoobservethe

sim-ilaritiesanddifferencesbetweentheorgansandtissuesofneotenic

andmetamorphosedAxolotl.

Conﬂictofintereststatement

Theauthorshavenoconﬂictsofinteresttodeclare.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,

intheonlineversion,athttp://dx.doi.org/10.1016/j.acthis.2016.07.

006.

References

Amamoto,R.,Huerta,V.G.,Takahashi,E.,Dai,G.,Grant,A.K.,Fu,Z.,Arlotta,P.,2016. Adultaxolotlscanregenerateoriginalneuronaldiversityinresponsetobrain injury.eLife5.

Atkinson,B.G.,Warkman,A.S.,Chen,Y.,1998.Thyroidhormoneinducesa reprogrammingofgeneexpressionintheliverofpremetamorphicRana catesbeianatadpoles.WoundRepairRegen.6(4),323–337.

Badawy,G.M.,2011.Effectofthyroidstimulatinghormoneontheultrastructureof thethyroidglandintheMexicanaxolotlduringmetamorphicclimax.J.Appl. Pharm.Sci.1(4),60–66.

Bertrand,S.,Brunet,F.G.,Escriva,H.,Parmentier,G.,Laudet,V.,Robinson-Rechavi, M.,2004.Evolutionarygenomicsofnuclearreceptors:fromtwenty-ﬁve ancestralgenestoderivedendocrinesystems.Mol.Biol.Evol.21(10), 1923–1937.

Billingham,R.E.,Silvers,W.K.,1967.Studiesontheconservationofepidermal speciﬁciesofskinandcertainmucosasinadultmammals.J.Exp.Med.125(3), 429–446.

Brown,D.D.,Cai,L.,2007.Amphibianmetamorphosis.Dev.Biol.306(1),20–33. Buchholz,D.R.,Paul,B.D.,Fu,L.,Shi,Y.B.,2006.Molecularanddevelopmental

analysesofthyroidhormonereceptorfunctioninXenopuslaevis,theAfrican clawedfrog.Gen.Comp.Endocrinol.145(1),1–19.

Burggren,W.W.,Warburton,S.,2007.Amphibiansasanimalmodelsforlaboratory researchinphysiology.ILARJ.48(3),260–269.

Colombo,B.M.,Scalvenzi,T.,Benlamara,S.,Pollet,N.,2015.Microbiotaand mucosalimmunityinamphibians.Front.Immunol.6,111.

Coots,P.S.,Seifert,A.W.,2015.Thyroxine-inducedmetamorphosisintheaxolotl (Ambystomamexicanum).MethodsMol.Biol.1290,141–145.

Cosden-Decker,R.S.,Bickett,M.M.,Lattermann,C.,MacLeod,J.N.,2012.Structural andfunctionalanalysisofintra-articularinterzonetissueinaxolotl

salamandersOsteoarthritisandcartilage/OARS.OsteoarthritisRes.Soc.20(11), 1347–1356.

Das,B.,Cai,L.,Carter,M.G.,Piao,Y.L.,Sharov,A.A.,Ko,M.S.,Brown,D.D.,2006.Gene expressionchangesatmetamorphosisinducedbythyroidhormoneinXenopus laevistadpoles.Dev.Biol.291(2),342–355.

Denis,J.F.,Levesque,M.,Tran,S.D.,Camarda,A.J.,Roy,S.,2013.Axolotlasamodel tostudyscarlesswoundhealinginvertebrates:roleofthetransforming growthfactorbetasignalingpathway.Adv.WoundCare2(5),250–260. Dierichs,R.,Dosche,C.,1982.Thealveolar-lininglayerinthelungoftheaxolotl,

Ambystomamexicanum.Anelectron-microscopicstudyusingheavymetal complexes.CellTissueRes.222(3),677–686.

Dodd,M.H.I.,Dodd,J.M.,1976.Thebiologyofmetamorphosis.In:Lofts,B.(Ed.), PhysiologyoftheAmphibia.AcademicPress,NewYork,pp.467–599. Eroschenko,V.P.,DiFiore,M.S.H.,2013.DiFiore’sAtlasofHistologywithFunctional

Correlations.LippincottWilliams&Wilkins.

Escriva,H.,Manzon,L.,Youson,J.,Laudet,V.,2002.Analysisoflampreyandhagﬁsh genesrevealsacomplexhistoryofgeneduplicationsduringearlyvertebrate evolution.Mol.Biol.Evol.19(9),1440–1450.

FriersonJr.,H.F.,1989.Thegrossanatomyandhistologyofthegallbladder, extrahepaticbileducts,Vateriansystem,andminorpapilla.Am.J.Surg.Pathol. 13(2),146–162.

Furlow,J.D.,Neff,E.S.,2006.Adevelopmentalswitchinducedbythyroidhormone: xenopuslaevismetamorphosis.ABBVTrendsEndocrinol.Metab.17(2),40–47. Galton,V.A.,1992.Thyroidhormonereceptorsandiodothyroninedeiodinasesin

thedevelopingMexicanaxolotl,Ambystomamexicanum.Gen.Comp. Endocrinol.85(1),62–70.

Hasebe,T.,Fu,L.,Miller,T.C.,Zhang,Y.,Shi,Y.B.,Ishizuya-Oka,A.,2013.Thyroid hormone-inducedcell–cellinteractionsarerequiredforthedevelopmentof adultintestinalstemcells.CellBiosci.3(1),18.

Hayes,T.,Chan,R.,Licht,P.,1993.Interactionsoftemperatureandsteroidson larvalgrowth,development,andmetamorphosisinatoad(Bufoboreas).J.Exp. Zool.266(3),206–215.

Huang,P.,Chandra,V.,Rastinejad,F.,2010.Structuraloverviewofthenuclear receptorsuperfamily:insightsintophysiologyandtherapeutics.Annu.Rev. Physiol.72,247–272.

Huggins,P.,Johnson,C.K.,Schoergendorfer,A.,Putta,S.,Bathke,A.C.,Stromberg, A.J.,Voss,S.R.,2012.Identiﬁcationofdifferentiallyexpressedthyroidhormone responsivegenesfromthebrainoftheMexicanAxolotl(Ambystoma mexicanum).Comparativebiochemistryandphysiology.Toxicol.Pharmacol. CBP155(1),128–135.

Hulbert,A.J.,2000.Thyroidhormonesandtheireffects:anewperspective.Biol. Rev.75(4),519–631.

Ishizuya-Oka,A.,Hasebe,T.,Shi,Y.B.,2010.Apoptosisinamphibianorgansduring metamorphosis.Apoptosis:Int.J.Program.CellDeath15(3),350–364. Jacobs,G.F.,Michielsen,R.P.,Kuhn,E.R.,1988.Thyroxineandtriiodothyroninein

plasmaandthyroidsoftheneotenicandmetamorphosedaxolotlAmbystoma mexicanum:inﬂuenceofTRHinjections.Gen.Comp.Endocrinol.70(1), 145–151.

Jonasson,K.A.,Russell,A.P.,Vickaryous,M.K.,2012.Histologyandhistochemistry ofthegekkotannotochordandtheirbearingonthedevelopmentof notochordalcartilage.J.Morphol.273(6),596–603.

Kliewer,S.A.,Umesono,K.,Mangelsdorf,D.J.,Evans,R.M.,1992.RetinoidXreceptor interactswithnuclearreceptorsinretinoicacid,thyroidhormoneandvitamin D3signalling.Nature355(6359),446–449.

Kragl,M.,Knapp,D.,Nacu,E.,Khattak,S.,Maden,M.,Epperlein,H.H.,Tanaka,E.M., 2009.Cellskeepamemoryoftheirtissueoriginduringaxolotllimb regeneration.Nature460(7251),60–65.

Kupferberg,S.J.,Marks,J.C.,Power,M.E.,1994.Effectsofvariationinnaturalalgal anddetritaldietsonlarvalanuran(Hyla-Regilla)life-historytraits.Copeia2, 446–457.

Lopez,D.,Lin,L.,Monaghan,J.R.,Cogle,C.R.,Bova,F.J.,Maden,M.,Scott,E.W.,2014. Mappinghematopoiesisinafullyregenerativevertebrate:theaxolotl.Blood 124(8),1232–1241.

Maden,M.,Manwell,L.A.,Ormerod,B.K.,2013.Proliferationzonesintheaxolotl brainandregenerationofthetelencephalon.NeuralDev.8,1.

Malvin,G.M.,Heisler,N.,1988.Bloodﬂowpatternsinthesalamander,Ambystoma tigrinum,before,duringandaftermetamorphosis.J.Exp.Biol.137,53–74. McCusker,C.,Gardiner,D.M.,2011.Theaxolotlmodelforregenerationandaging

research:amini-review.Gerontology57(6),565–571.

Menger,B.,Vogt,P.M.,Jacobsen,I.D.,Allmeling,C.,Kuhbier,J.W.,Mutschmann,F., Reimers,K.,2010.Resectionofalargeintra-abdominaltumorintheMexican axolotl:acasereport.Vet.Surg.:VS39(2),232–233.

Monaghan,J.R.,Stier,A.C.,Michonneau,F.,Smith,M.D.,Pasch,B.,Maden,M., Seifert,A.W.,2014.Experimentallyinducedmetamorphosisinaxolotlsreduces regenerativerateandﬁdelity.Regeneration1(1),2–14.

Mondou,P.M.,Kaltenbach,J.C.,1979.Thyroxineconcentrationsinbloodserumand pericardialﬂuidofmetamorphosingtadpolesandofadultfrogs.Gen.Comp. Endocrinol.39(3),343–349.

Nussey,S.,Whitehead,S.,2013.Endocrinology:anIntegratedApproach.CRCPress. Page,R.B.,Voss,S.R.,2009.InductionofMetamorphosisinAxolotls(Ambystoma

Mexicanum).ColdSpringHarborprotocols(pdbprot5268).

Page,R.B.,Monaghan,J.R.,Walker,J.A.,Voss,S.R.,2009.Amodeloftranscriptional andmorphologicalchangesduringthyroidhormone-inducedmetamorphosis oftheaxolotl.Gen.Comp.Endocrinol.162(2),219–232.

Paris,M.,Laudet,V.,2008.Thehistoryofadevelopmentalstage:metamorphosisin chordates.Genesis46(11),657–672.

Rajguru,J.,Jain,S.,Khare,S.,Fulzele,R.R.,Ghai,R.,2013.Embryologicalbasisand clinicalcorrelationoftherarecongenitalanomalyofthehumangallbladder:– thediverticulum–amorphologicalstudy.J.Clin.Diagn.Res.:JCDR7(10), 2107–2110.

RodrigoAlbors,A.,Tazaki,A.,Rost,F.,Nowoshilow,S.,Chara,O.,Tanaka,E.M.,2015. Planarcellpolarity-mediatedinductionofneuralstemcellexpansionduring axolotlspinalcordregeneration.eLife4.

Rosenkilde,P.,Ussing,A.P.,1996.Whatmechanismscontrolneotenyandregulate inducedmetamorphosisinurodeles?Int.J.Dev.Biol.40(4),665–673. Rovira,J.,Villaro,A.C.,Bodegas,M.E.,Valverde,E.,Sesma,P.,1995.Metamorphic

changesinthestomachofthefrogRanatemporariatadpoles.Tissuecell27(1), 13–22.

Satoh,A.,Mitogawa,K.,Makanae,A.,2015.Regenerationinducersinlimb regeneration.Dev.GrowthDiffer.57(6),421–429.

Schnapp,E.,Kragl,M.,Rubin,L.,Tanaka,E.M.,2005.Hedgehogsignalingcontrols dorsoventralpatterning,blastemacellproliferationandcartilageinduction duringaxolotltailregeneration.Development132(14),3243–3253. Seifert,A.W.,Monaghan,J.R.,Voss,S.R.,Maden,M.,2012.Skinregenerationinadult

axolotls:ablueprintforscar-freehealinginvertebrates.PLoSOne7(4), e32875.

Semlitsch,R.D.,Caldwell,J.P.,1982.Effectsofdensityongrowth,metamorphosis, andsurvivorshipintadpolesofScaphiopus-Holbrooki.Ecology63(4), 905–911.

Shi,Y.B.,2000.Amphibianmetamorphosis.In:FromMorphologytoMolecular Biology.Wiley-Liss,NewYork,pp.p288.

(14)