myeloproliferation7788kk.com

REVIEWSpecicationandfunctionofhemogenicendotheliumduringembryogenesisEmilyGritz1,2KarenK.
Hirschi1Received:20August2015/Revised:16December2015/Accepted:7January2016/Publishedonline:5February2016TheAuthor(s)2016.
ThisarticleispublishedwithopenaccessatSpringerlink.
comAbstractHemogenicendotheliumisaspecializedsubsetofdevelopingvascularendotheliumthatacquireshematopoieticpotentialandcangiverisetomultilineagehematopoieticstemandprogenitorcellsduringanarrowdevelopmentalwindowintissuessuchastheextraembry-onicyolksacandembryonicaorta-gonad-mesonephros.
Herein,wereviewcurrentknowledgeaboutthehistoricalanddevelopmentaloriginsofhemogenicendothelium,themoleculareventsthatgovernhemogenicspecicationofvascularendothelialcells,thegenerationofmultilineagehematopoieticstemandprogenitorcellsfromhemogenicendothelium,andthepotentialfortranslationalapplica-tionsofknowledgegainedfromfurtherstudyoftheseprocesses.
keywordsEmbryogenesisDevelopmentalhematopoiesisHemogenicendotheliumEndothelialtohematopoietictransitionIntroductionNormalembryogenesisispredicateduponthedevelopmentofafunctioningcardiovascularsystemearlyingestation—disruptionsinthisleadtovascular,cardiac,hematologicdisease,andinextremecasesembryoniclethality.
Astheprimitivevasculatureexpandsthroughoutearlydevelop-ment,itremodelsandspecializestofulllthecriticalmetabolicdemandsofthetissuesitsupplies[1].
Aspartofthisremodeling,endothelialcellsliningthecardiovascularsystemsubspecializetoarterial,venous,lymphatic,andhemogenicfatesviaacomplexnetworkofintersectingmolecularpathways.
Themechanismsunderlyingnormalvasculardevelopmentremainasyetincompletelydened,andagreaterunderstandingoftheseprocesseswillenhanceourunderstandingofcertainpathologicstatesrelatedtothevascularsystemandhowtheymightbetreated.
Hemogenicendotheliumisasmall,specializedsubsetofvascularendotheliumthatacquireshematopoieticpotentialandcangiverisetomultilineagehematopoieticstemandprogenitorcells(HSPC)withinanarrowdevelopmentalwindowwithindistincttissues[1].
Workinthelastdecadehasbeguntoclarifythecomplexsignalsthatleadtoacquisitionofthisblood-formingpotential.
Ourunder-standingoftheeventsthatleadtohemogenicspecicationofendothelialcellsfromvascularendothelium,aswellastheeventsthatleadtogenerationofHSPCfromhemogenicendotheliumisstillinitsinfancy.
Thisreviewwilloutlinecurrentknowledgeaboutthehistoricalanddevelopmentaloriginsofhemogenicendothelium,themoleculareventsthatgovernhemogenicspecicationofvascularendothelialcells,thegenerationofmultilineageHSPCfromhemogenicendothelium,andthepotentialforclini-cal/translationalapplicationsofknowledgegainedfromfurtherstudyoftheseprocesses.
&KarenK.
Hirschikaren.
hirschi@yale.
edu1DepartmentsofMedicine,GeneticsandBiomedicalEngineering,YaleCardiovascularResearchCenter,VascularBiologyandTherapeuticsProgram,andYaleStemCellCenter,YaleUniversitySchoolofMedicine,300GeorgeSt.
,NewHaven,CT06511,USA2DepartmentofPediatrics,SectionofNeonatal-PerinatalMedicine,YaleUniversitySchoolofMedicine,333CedarSt.
,NewHaven,CT06511,USACell.
Mol.
LifeSci.
(2016)73:1547–1567DOI10.
1007/s00018-016-2134-0CellularandMolecularLifeSciences123DevelopmentalandhistoricaloriginsofhemogenicendotheliumPrimitivehematopoiesisHematopoiesisoccursintwowavesduringembryonicdevelopment—theprimitive(precirculation)waveanddenitivewave.
Therstbloodandendothelialcellsarederivedfromtheextraembryonicmesodermbeginningaroundembryonicday(E)7.
0[2];whethertheyarederivedfromacommonbipotentprogenitor(hemangioblast),orareindependentlyfated,remainsasubjectofdebate,asdis-cussedlaterinthisreview.
Primitivehematopoiesisismarkedbythebriefemergenceofmostlynucleatedery-throidprogenitors,aswellasmacrophageprogenitors[3],thataregeneratedinparallelwithendothelialcells.
Theseprimitiveerythroidcellsarefunctionallyandmorphologi-callydistinctfromlatererythroidprogenitorsinthattheyarelarge,nucleated,expressembryonicglobins,andareexclusivelydetectedintheextra-embyonicyolksacduringanarrow(*48h)windowbeforetheydisappear[3].
Otherprecirculationextra-embryonicsourcesofpro-denitiveerythroidandmyeloidprogenitorcellslateremergebetweenE7.
5andE8.
0andincludetheallantois[4,5]andpara-aorticsplanchnopleura(pSP)[6,7].
Theseearlycellsareofgreatimportancetothedevelopingembryoastheysustainembryonicsurvivaluntilthedenitivehematopoieticsystemisestablished,andtheyformstructuresreferredtoas''bloodislands''aroundE7.
5[8],inwhichthecellsintheinteriorgiverisetonucleatederythroidandmyeloidcellsandthoseontheperipherybecomelumenizedendothelialcells[1].
Bloodislandsthencoalescetoformvascularchannelsandaplexusnetworkthroughouttheyolksac,whichconductsoscillatoryplasmaowdrivenbythedevelopinghearttube[9].
ByE8.
25,asecondwaveoferythromyeloidprogenitor(EMP)cellsaregeneratedwhichalsogiverisetotissuemacrophagesthatpersistintoadulthood[10–12].
Followingtheonsetofplasmaow,theimmatureprogenitorcellsjointheplasmacirculation[3,13].
DenitivehematopoiesisDenitivehematopoiesisisthesecondphaseofbloodproductionbeginningwithproductionoferythroidandmyeloidprogenitorsthatisthenfollowedbygenerationoflong-termrepopulatinghematopoieticstemcells(HSC)capableofcolonizinghematopoieticorgansandgivingrisetoalldifferentiatedbloodlineages[14,15].
DenitivehematopoiesiscoincideswiththeonsetofpulsatilebloodowduetocoordinatedcardiaccontractionbeginningaroundE8.
25,astherstwaveofextra-embryonicprimitiveerythroidprogenitorspreviouslydescribeddecreaseinnumberandthepreviouslydescribedsecondyolksacwaveoferythroidandmyeloidcellsaregeneratedandentertheplasmacirculation[3,14,16].
GenerationofhematopoieticprogenitorsthenbeginsintheplacentabetweenE9.
0and9.
5[17–20].
Inthemurineembryoproper,denitivehematopoiesisoccurslater,withintheaorta-gonad-mesonephros(AGM)regionpeakingatE10.
5[21–23]andismarkedbythegenerationofbothprogenitorcells,aswellasHSC.
TheseAGMandyolk-sac-derivedhematopoieticstemandprogenitorcellsarethoughttomigrateto,andcolonizeothersitesofdenitivehemato-poiesis,mostnotablythefetalliverbyE11.
0–E12.
0[24–26]andthebonemarrowatE16.
5[27],andcontributetotheadultHSCpool.
Whileitisclearthatprogenitorcellsemergefromtheyolksacandplacentaltissuesduringdenitivehematopoiesis,itremainsdebatedwhetherornottheplacentaandyolksacactuallygenerateHSCde-novo—althoughithasbeenshownthatthenumberofHSCcol-onizingthefetalliverisgreaterthanwhatcanbeaccountedforbytheAGMalone[26].
ThissuggeststhatadditionalsourcesofHSC,suchastheplacentaandyolksac,mayexistandcontributetotheHSCpoolusedbytheliverforself-expansion[14,18,26,28].
Italsoremainsunclearwhetherthereexistsde-novogenerationofHSCwithinfetalliver,fetalbonemarroworpostnataltissues,orifHSCaremerelyreplenishedviaself-renewalofyolksac-andAGM-derivedcells[29].
WhileithasbeendemonstratedthattheyolksacandAGMaresitesofdenitivehematopoiesis,thecellularoriginsoftheHSPCwithinthesetissuesisstillunderinvestigation,andmaybedifferentindistinctspecies.
Nonetheless,itisgenerallythoughtthatmurinedenitiveHSPCarederivedfromasmallsubsetofvascularendotheliumthatacquireshemogenicpotential,so-calledhemogenicendothelium.
Interestingly,hematopoieticcellsproducedbyhemogenicendothelialcellshavedifferentfunctionalcharacteristicsdependingontheirsiteoforigin[29].
Thatis,yolksachemogenicendothelialcellsgiverisetomultilineagehematopoieticprogenitorsthatcannotrepopulateneonataloradultbonemarrowlong-terminvivo[7],andarethereforereferredtoasmultilineageprogenitors.
Incontrast,AGM-derivedhematopoieticcellsarecapableofrescuingirradiatedneonatalandadultrecipients[30];thus,thesecellsareconsidereddenitiveHSC.
Thisraisesthepossibilitythatnotallhemogenicendothelialcellsarecreatedequally,orthattheiranatomicnicheinuencestheirfunctionandprogeny,inamannerdependentonthedevelopmentaltimingoftheiremergenceand/orthecellularandextracellularcompositionoftheirmicroenvironment[29].
1548E.
Gritz,K.
K.
Hirschi123ConceptevolutionofhemogenicendotheliumTobestunderstandthesignicanceofhemogenicendothelialcellsinembryonichematopoiesis,itisusefultoconsidertheevolutionoftheconceptoftheirexistenceandfunctionoverthecourseofthelastcentury.
Insightsgainedduringtheirdiscovery,identicationandcharacterizationhavehelpedintertwinetheeldsofhematopoieticstemcellbiologyandvascularbiology.
Theconceptofhemogenicendotheliumdatesbackinthescienticliteraturetothelate19thcentury.
Beginningin1899,severalanatomistsobservedanassociationofbloodformingcellswithdevelopingvascularstructuresacrossawidevarietyofmammalianspeciesincludingthebat(1899),chick(1907),rabbit(1909),human(1912),pig(1916),andmongoose(1917)[15].
In1917,anatomistFlorenceSabindirectlyobservedinchickembryostudiesthat''redblood-corpus-clescanbeseentogrowfromtheendothelialliningofblood-vessels''[31].
Thesebloodcellswerethennotedtobudofffromvascularendothelialcellsintoplasmaowingthroughprimitivevascularplexusofthedevelopingchick.
Inthe1930s,Murrayre-brandedSabin's''angioblast''asa''hemangioblast''orasubsetofmesenchymally-derivedprimitiveendotheliumthattransientlyacquiresblood-formingpotential[32].
Overthenextseveraldec-ades,however,thisconceptofthehemangioblastandthequestionssurroundinganassociationbetweenvascularendotheliumandbloodformationwentlargelyunad-dressed.
Inthe1960s,MooreandOwensuggestedthatallembryonichematopoiesisoccurredintheextra-embryonicyolksac,andthatintra-embryonicbloodinlaterstagesofdevelopmentwasaresultofmigrationofyolksacderivedhematopoieticprogenitors[15].
Thisconceptofexclu-sivelyextra-embryonichematopoiesiswasrefutedbyDieterlen-Lie`vreandcoworkersin1975whentheydemonstratedintraembryonicaortichemogenesisinquail-chickembryograftingexperimentsandrenewedinterestintheoriginofhematopoieticstemandprogenitorcellsinthedevelopingembryo[15,33].
ThehemangioblastOverthenexttwodecades,severalgroupscorroboratedtheworkofDieterlen-Lie`vreandcoworkersbydemonstratingthepresenceofhematopoieticstemandprogenitorcellsintheventralaspectofthedevelopingdorsalaortapriortohepaticcolonization,anddemonstratedthatthesecellclustersarenecessaryfortheestablishmentofdenitivehematopoiesis[6,15,23,24,34,35].
Thisconrmedassociationofhematopoieticcellswithvascularendothe-liumbroughtintoquestionthedevelopmentalrelationshipbetweenhematopoieticcellsandvascularendothelium—weretheytwoindependentlineagesderivedfromdiscreteprogenitorcelltypesordidtheyhaveacommonmeso-dermally-derivedbipotentcellprogenitor,theso-calledhemangioblast,thattransientlygivesrisetovascularendothelialandbloodcells[29].
Indeed,endothelialandhematopoieticlineageshavebeenshowntosharemanycommonsurfacemarkersandtranscriptionfactorsthatareimplicatedthroughouttheirdifferentiation.
TheseincludemesodermalmarkersBrachyury(Bry),bonemorphogenicprotein4(BMP4),andvascularendothelialgrowthfactor2(VEGFR2orFlk-1)[36];sharedmarkersCD34,VE-cad-herin,andCD31;andcommontranscriptionfactorsRUNX1andGATAbindingprotein2(GATA2)[37],lendingcredibilitytothehemangioblastconcept.
However,todate,thereisalackofconvincingevidencetosuggestthatsuchanexclusivelybipotentmesodermalprogenitorexists[29].
Thebipotenthemangioblasthasbeendescribedasco-expressingmesodermalmarkerBryandFlk-1,butBryFlk-1cellshave,infact,beendemonstratedtogiverisetocardiac,skeletal,andvascularsmoothmuscle,aswellasendothelialcellsandbloodcells[38,39].
Padron-Bartheandcoworkersdemonstratedbyinvivoclonalanalysisofmurineembryosthattheearliestbloodandendotheliallineagesdonotarisefromaprimitivestreak-derivedbipotentialprecursor,butthattheselineagesarederivedfromindependentepiblastpopulations[40].
Theseresultsareconsistentwithpreviousmesodermalgraftingstudiesthatalsosuggestendothelialandbloodcelllineagesareindependentlyfatedduringprimitivehematopoiesis[41].
Itremainspossiblethattheremayexistabipotentmesodermalprogenitorgivingrisetoexclusivelyendothelialandbloodcells,butthishasnotbeendeni-tivelydemonstratedtodate[29].
AmiddlegroundtothehemangioblastdebatewasproposedbyLancrinandcoworkers,whereinthemesodermally-derivedheman-gioblastgivesrisetoanendothelialintermediate(hemogenicendothelium)thatthengivesrisetoHSPC[42].
HemogenicendotheliumDespitetheuncertaintyoftheexistenceofhemangioblastsduringprimitivehematopoiesis,severallandmarkstudiesoverthelasttwodecadeshaveprovidedstrongevidencethatduringdenitivehematopoiesisinvertebrates,multi-potentHSPCarisedirectlyfromtransientlyspecializedvascularendothelium(hemogenicendothelium)withinthedevelopingAGMbetweenE10.
0andE11.
5,priortoappearanceinotherintra-embryonichematopoieticorgans[6,23,24,34,35].
Theaforementionedearlyobservationsofbloodcellsbuddingfromvascularendotheliumhavebeencorrobo-ratedbydye-labeledfatetracingapproachesthatSpecicationandfunctionofhemogenicendotheliumduringembryogenesis1549123demonstrateacrossmultipleanimalmodelsthatdye-taggedvascularendotheliuminthedorsalaortagivesrisetodye-taggedcirculatingbloodcells[43,44].
SubsequentinvivofatetracingstudiesinmurineembryoshasfurtherdemonstratedemergenceofHSCfromendotheliumfromVE-cadcellsinthedorsalaortathatgoontocolonizeintra-embryonichematopoieticsitesincludingthebonemarrow,spleen,andthymus[45].
Hematopoieticstemand/orprogenitorcellemergencehasalsobeenshowntooccurwithinthevascularplexiwithintheextra-embryonicyolksac[13,46,47],placenta[18],aswellasthevitellineartery,umbilicalarteries[21,48,49],endocardium[50],andheadarteries[51].
Multiplereal-timeimagingstudiesdemonstratinghematopoieticcellemergencefromvascularendotheliumhavebeenperformedinmiceandzebrash[52–54].
SinglecelllineagetracingstudieshavedemonstratedmultipotentHSPCwithcellsurfacemarkersCD31(endothelialcellmarker),CD41(bloodcellmarker),c-Kit(stemcellgrowthfactorrecep-tor),andSCA-1(stemcellantigen,alsoknownasLy6A)arisingdirectlyfromtheventralwallofthemurinedorsalaortaethatcanbetracedtothefetalliver[53],andulti-matelytotheadulthematopoieticsysteminmice[45]andinzebrash[52,54].
Severalstudieshaveaddressedthequestionofwhethervascular-derivedhematopoiesistrulyoriginatesfromendothelialcellsandisnotmesenchymalinorigin.
In2002,deBruijnandcolleaguesdemonstratedusingaLy6A(SCA-1)-GFPtransgenicmousemodelthatallhematopoieticstemcellswithinthedevelopingaortaareLy6A-GFPandthatthesecellslocalizetotheendotheliallayerofthedorsalaorta,butnottotheunderlyingmesenchyme[55].
TofurthersupporttheassertionthatHSPCaregeneratedfromanendothelialintermediateandnottheunderlyingmesenchyme,ithasbeendemonstratedthatkeyhematopoieticregulatorssuchasthetranscriptionfactorRUNX1areexpressedathighlevelsbyendothelialcellsinhemogenicvascularsitesinvertebrates[56].
NorthandcolleaguesfurtherdemonstratedthatwhileRUNX1-ex-pressingHSCarefoundinbothendothelialandmesenchymalcellfractionsinthemouseembryo,thepresenceoftwofunctionalRunx1allelessegregatesHSCtotheendothelialfractiononly[57].
Morerecently,ithasbeenshownthatRunx1deletioninVE-cadherinexpressingendothelialcellsresultsinlossofintra-aortichematopoi-eticclusterformation,againcorroboratingthelinkbetweencellsofendothelialorigingivingrisetohematopoieticclustersviatheactionofknownhematopoieticmediatorssuchasRUNX1[58].
Additionally,murineAGM-associ-atedhemogenicendothelialcellsgiverisetoeitherhematopoieticorendotheliallineagesbutneverboth[59]andhumanpluripotentstemcell-derivedarterialendothe-lialcellsandhemogenicendothelialcellscanbedistinguishedonthebasisofCD184andCD73expression[60].
Thesestudiescollectivelydemonstrate,onafunctionalaswellasmorphologicallevel,astrongbodyofevidencefortheexistenceofhemogenicendothelialcellsofexclu-sivelyvascularoriginthatgiverisetomultilineageHSPCduringdenitivehematopoiesis.
However,muchlessisknownaboutthemechanismsunderlyinghemogenicspecicationandtheirsubsequentgenerationofHSPC.
CharacterizationofhemogenicendotheliumInordertocharacterizethemoleculareventsunderlyinghemogenicendothelialcellspecicationandtheirsubse-quentgenerationofHSPC,itisnecessarytodelineatethephenotypeofhemogenicvs.
non-bloodformingendothe-lium,sothattheycanbeeffectivelyisolatedandstudied.
Isolationofthesecellsprovestobeadifcultundertakingashemogenicendotheliumrepresentsasmall(*1–3%ofmurineyolksacandAGMendothelialcells)andtransientpopulationwithinhematopoietictissues[13,61].
Todate,nodenitivesinglemarkertodistinguishhemogenicfromnonhemogenicendothelialcellshasbeenidentied.
Buildinguponthebodyofevidencesupportingtheveryexistenceofhemogenicendothelium,severalgroupshaveusedowcytometrytechniquestoelucidatethephenotypicidentityofhemogenicendothelialcellswithintheyolksacandAGMtoaidintheirisolationandfurtherstudy.
In1997,KabrunandcolleaguesdescribedgenerationofHSPCfromFlk1endothelium[62].
Shortlythereafter,NishikawaandcolleaguesisolatedVE-cadCD45-Ter119-cellsfromtheyolksacandcaudalhalfofembryoproperofE9.
5mouseembryosanddemonstratedthatthiscellfractionwascapableofgivingrisetolymphohematopoieticcellsinculture.
ThisstudyconrmedthattheVE-cadcellfractionco-expressedvascularmarkersCD34,CD31,andFlk-1,conrmingthattheisolatedcellswereindeedendothelialinoriginandconrmingboththeexistenceandfunctionalcapabilityofhemogenicendotheliumtogiverisetobloodprogenitors[63].
TheneedforVE-cadherinexpressionwasfurthercorroboratedbyasubsequentstudybyFraserandcol-leaguesthatdemonstratedlongtermlyphohematopoieticreconstitutionpotentialofVE-cad/CD45-cellsinjectedintoirradiatedneonatalmouserecipients[30].
Hemogenicendothelialcellscanalsobedistinguishedfromnon-hemogenicendothelialcellsbasedondifferentialactivityofaregulatoryelementofaKDRpromoterenhancersuch1550E.
Gritz,K.
K.
Hirschi123thatonlynonhemogenicendothelialcellsactivatetheenhancer[64].
Otherstudieshavesinceshownthatcellsco-expressingCD31,CD34,andFlk-1frombothmurineandhumanyolksacandAGMhavedemonstratedlympho-andlymphomyelopoieticpotentialundercultureconditions[1,63,65].
Inaddition,cellsco-expressingFlk-1andVE-cadherinexhibitenhancedcolonyformingabilitywhencomparedtoFlk-1VE-cad-cells[30];however,VE-cadherinexpressionwasnotfoundtobenecessaryfortheblood-formingabilityofyolksachemogenicendothelialcellsinmice[13]orfortransitionofhemogenicendothelialcellstoHSCinzebrashandmice[66].
Ithasbeenshownaswellthathemogenicendothelialcellsmayactuallybedividedintofunctionallydistinctpopulationsthateithergenerateerythroidandmyeloidprogenitorcellsorexclu-sivelygenerateHSC[67].
Alpha4-integrin(a4-integrin)hasbeenidentiedasamarkerthatdistinguisheshemogenicVE-cadcellsfromnonhemogenicVE-cadcells[68].
Livecelltime-lapsedimagingofembryonicstemcell-derivedFlk1VE-cad-mesodermalcellsco-culturedwithOP9stromalcellsdemonstratedtransformationofVE-cadDI-acylLDLcellswithClaudin5mediated-tightjunctionsintosheetsofcellswithendothelialmorphologythatgaverisetonon-adherent,free-oatingroundCD45CD41he-matopoieticprogenitorcells[69].
Lossofendothelialmarkersandacquisitionofhematopoieticmarkerswasshowntoproceedinapatternedfashioninthismodel.
Morerecently,acriticalroleforStemcellleukemia(SCL)factorinmaintenanceofhemogeniccompetenceandpreventionofcardiomyocyteprogrammingofprimitivevascularendothelialcellshasbeenidentied[70].
Inotherrecentstudies,singlehemogenicandnon-hemogenicendothelialcellscouldbeisolatedfromRunx1enhancer-reportermice,whichwasbenecialfortranscriptionalprolingofthesecelltypes[59,69].
Furtherphenotypiccharacterizationofhemogenicendothelialcellsbyourgrouphasrevealedthatblood-formingactivityofbothyolksacandAGMtissuesiscon-tainedwithintheHoechstdyeefuxing,or''sidepopulation''(SP)fractionofcells[13,61,71],whichisnotsurprisinggiventhattheSPphenotypeisalsoacharacter-isticofHSCwithinadultbonemarrow[72].
SPcellswithintheyolksacandAGMwerefurtherfractionatedbasedonendothelialandhematopoieticcellsurfacemarkers,andthephenotypeofhemogenicendotheliumthereinhasbeendened,onaclonallevel,asFlk-1c-KitCD45-SPcells[13,61,71].
Inaddition,theFlk-1c-KitCD45-SPhemogenicendothelialcellsweredemonstratedtogiverisetomultilineageHSPCthatareFlk1-c-KitCD45SPcells[13],thatcanbedistinguishedfrommaturebloodcelltypeswithaFlk1-CD31±CD45non-SPphenotype.
SpecicationofhemogenicendotheliumDespitetheseknowledgegainsaboutthecharacteristicsofhemogenicendotheliumoverthelastdecade,littleisknownaboutthehierarchyofmoleculareventsthatgoverntheirspecicationfromnon-bloodformingendothelium,aswellastheeventsthatleadtotheirgenerationofHSPC.
However,thisbodyofknowledgeisgrowingandrecentadvancesinourunderstandingarediscussedbelow(andaresummarizedinFig.
1).
RetinoicacidsignalingRetinoicacid(RA)isknowntoregulatemurineendothelialcelldevelopmentandspecication[73,74].
Biologicallyactiveretinoicacid,knownasall-transretinoicacid(ATRA),isderivedfromretinol(VitaminA)viaoxidationbyretinaldehydedehydrogenases(RALDH1-3).
ATRAisthenreleased,takenupbytargetcells,andboundtomembersoftheretinoicacidreceptor(RAR)family(a,b,orc).
HeterodimerizationofRARfamilymemberswithrexinoidreceptors(RXR)a,b,andcformactivetran-scriptionfactorcomplexesthatbindtoretinoicacidresponseelementswithintargetgenestoinitiatetran-scription(reviewedinMarcelo[1]andChanda[75]).
Duringvasculogenesisinthemurineyolksac,activeRAisgeneratedbyRALDH2expressedinthevisceralendo-derm,whichthensignalswithinendothelialcellsintheadjacentmesodermthatselectivelyexpressRARa-1anda-2[73].
Raldh2-/-mutantembryosexhibitabnormalvasculardevelopment,duetohyperproliferativeendothe-lialcells[74],aswellasanemia,anddiearoundE10.
0[76].
OtherworkhasdemonstratedthatmicedecientforRAreceptorshaveimpairedfetalhepaticerythropoiesis[77]aswellasimpairedgenerationofmarrow-derivedHSC[78].
Becauseofitsessentialroleinendothelialcelldevelopment,aswellasembryonicandpostnatalhemato-poiesis,RAsignalingisofgreatinterestinthestudyofspecicationofendothelialcellstoahemogenicstate.
AlthoughRaldh1andRaldh3nullembryoshavenotbeenshowntoexhibithematologicdefects[79],embryoslackingRaldh2wereshowntoexhibitdefectsindenitive,butnotprimitivehematopoiesis[13,75].
Inaddition,wefoundthattheunderlyinghematopoieticdefectinRald-h2-/-mutantsislackofhemogenicendothelialcelldevelopment,whichcouldberescuedviaprovisionofbioactiveRAtoRaldh2-/-embryosinuteroorinculture[13,61].
RAsignalingiscriticalforthedevelopmentofhemogenicendothelialcellswithintheyolksac,aswellasAGM.
Inbothtissues,*90%ofendothelialcellswithactiveRAsignalingexhibitahemogenicphenotype;inaddition,*90%ofhemogenicendothelialcellsareSpecicationandfunctionofhemogenicendotheliumduringembryogenesis1551123undergoingactiveRAsignalingduringdenitivehemato-poiesisinthesetissues[61,75].
ThesendingsplaceRAatorverynearthetopofthesignalinghierarchygoverninghemogenicendothelialcellspecicationandfunctionduringdenitivehematopoiesis[13].
NotchTheNotchsignalingpathwayisevolutionarilyconservedandinvolvedinmanyaspectsofembryonicdevelopment,withnotablerolesinvascularandcardiacdevelopment.
MammalsexpressfourmembersoftheNotchfamilyofreceptors(Notch1-4)andveligands:Delta-like1,3,4(DLL)andJagged1and2(Jag).
TheinteractionsbetweenthereceptorsandligandsallowsignaltransductionbetweenneighboringcellsviaamultistepproteolyticcleavageoftheNotchreceptorandsubsequentreleaseoftheNotchintracellulardomain(NICD)(reviewedinHigh2008[80]).
Ac-secretasecomplexpowersthenalstepofreleaseofNICDintothecytoplasm,whereinNICDcanthenenterthenucleustoformactivetranscriptionalcomplexeswithRBPJ,andco-activatorMastermind-like(MAML)[80].
Thepathwayitselfappearsstraightforward,intheory,butisregulatedbyseveralpositiveandnegativefeedbackloopswhoseactionsaredeterminedbybothcellularcon-text,developmentaltiming,andlocalenvironment[80].
Notchsignalingiswellknowntobeinvolvedincellfatedecisionsandcelldifferentiation,particularlyinendothe-lialandbloodcells,andagrowingbodyofevidencealsopointstowarditsinvolvementinhemogenicspecication.
EmbryoslackingNotch1orNotch1andNotch4receptors,theonlyreceptorsexpressedbyendothelialcells[81,82],exhibitabnormalvasculardevelopmentsimilartoRaldh2-/-mutants[74,83,84],andtheexpressionofNotch1,anditsdownstreameffectorsHES1andHEY1,whichareknowntoregulatehematopoiesis[85]areupregulatedinendothelialcellsbyRA[61].
Inaddition,chemicalinhi-bitionofNotchsignalinginE8.
0wild-typemouseembryosviauseofc-secretaseinhibitorDAPTsuppressesyolksachemogenicendothelialcellspecication[61].
Notch1,specically,isexpressedintheventralwallofthedorsalaortawherehemogenicendotheliumforms(reviewedinJang2015[86]),andtheAGMofNotch1-/-mutantsexhibitdecreasedhematopoieticactivity(reviewedinZape2011[37,87]).
Conversely,Notch1inductioninmurineembryonicstemcellsleadstoanexpansionofVE-cadhemogenicendothelialcellswithenhancedhematopoieticpotential[86].
Thesedatacollectivelysug-gestthatNotch1receptorsignalingmayregulatehemogenicendothelialcellspecicationandfunction,andalsoplaceNotchdownstreamofRAinthesignalinghierarchygoverninghemogenicspecication.
c-Kitc-Kit(CD117)isagrowthfactorreceptorwithtyrosinekinaseactivityknowntobindstemcellfactor(SCF),whichleadstodimerizationofreceptorsandsubsequentactiva-tionorregulationofkinaseactivitythatmodulatesintracellularsignaltransductionpathwaysinvolvedincel-lularproliferation,maintenanceandmigration[1].
SuchpathwaysincludeSrckinase,PI3K,JAK-STAT,MAPK,andPLCc,aswellasregulatoryPTPases,phosphatidyli-nositolphosphatases,andproteinkinaseC[88].
SCFisknowntosupportmultilineagehematopoieticdevelopment[88]andc-KitmutationsareassociatedwithembryonicFig.
1Summaryofregulationofprimitivehematopoieticspecica-tionandgenerationofhematopoieticstemcellsduringembryonicdevelopment.
aSchematicrepresentationofendotheliallayerofdevelopingvascularwall(pink),listedareintra-andextraembryonicsourcesofvascularendothelialcellswithhemogenicpotential(bluebox).
bSchematicrepresentationofprogressionofeventsandmolecularsignalsgoverninghemogenicspecication(orangeboxes)andcendothelialtohematopoietictransition(greenboxes),endingwithgenerationofintra-vascularhematopoieticclustersandmulti-lineagehematopoieticstemandprogenitorcells(HSPC)1552E.
Gritz,K.
K.
Hirschi123lethalityatmidgestation,anemiaanddisruptedHSCdevelopment[88,89].
Aspreviouslynoted,theexpressionofc-Kitisadistinguishingfeatureofhemogenicendothelialcells,relativetonon-bloodformingendothelialcells.
Inaddition,Notch1expressioninendothelialcellsisupregulateddownstreamofc-Kit;thus,thispathwayappearstoplayacritical,yetstillundenedrole,inthesignalinghierarchythatgovernshemogenicendothelialcellspecication[61].
Cellcyclecontrol/p27Retinoicacid-decientandNotch-inhibitedembryossimi-larlyexhibitendothelialcellhyper-proliferation,andimpairedhemogenicendothelialcelldevelopment.
Wefoundthatdownregulationofcellcycleinhibitorp27,whichinducesG1arrest[73,74],tobetheunderlyingdefectinRA-decientembryos.
Furthermore,p27isupregulateddownstreamofNotchandc-Kit,andlentivirusmediatedre-expressionofp27inbothRA-andNotch-inhibitedmurineyolksacendothelialcellsrescuesendothelialcellcyclecontrol,hemogenicspecication,andthegenerationofHSPC[61].
GiventhatcellcyclestateanddurationofG1phasehavebeenshowntobedeter-minantsofstemcellfate[90–92],itispossiblethatendothelialcellsmustbearrestedinG1phaseinordertoundergohemogenicspecication.
Infact,severaltran-scriptionalregulatorsofdenitivehematopoiesisaredifferentiallyexpressedinG1phase(reviewedextensivelyin[93]).
Itisalsopossiblethatp27functionsasatran-scriptionalregulatortodirectlyalterendothelialcellphenotype,giventhatitcanrepressmultipotencygenesinstemcellpopulations[94].
Thus,theRA/c-Kit/Notchsig-nalingaxiswhichmediatesp27expressionmayregulatehemogenicspecicationinatleasttwoways:(1)viarepressionofgenesthatmaintainamultipotentstateinprimordialendothelialcellsorinductionofhemogenicgenes;and/or(2)elongationofG1phaseofcellcycletoenableexpressionandaccumulationoftranscriptionalregulatorsthatalterendothelialcellphenotype[61].
Generationofhematopoieticstem/progenitorcellsfromhemogenicendotheliumTheprocessbywhichHSCaregeneratedfromhemogenicendotheliumisreferredtoastheendothelial-to-he-matopoietictransition(EHT;depictedinFig.
1).
InordertobeabletoderiveHSCfrompluripotentstemcellsforclinicalandresearchapplications,anunderstandingoftheeventsthatleadtoHSCformationfromendothelialcellsinvivoisrequired.
TheeventsleadingtoHSCgenerationfromanendothelialintermediateatmultipleanatomicsites,includingtheyolksac,placenta,andAGM,involvesanincompletelycharacterizedcomplexinterplaybetweenmicroenvironment,developmentaltiming,andtranscrip-tionalregulationoffate.
TheAGMasasourceofHSCPriortotheonsetofcirculation,ithasbeenshownthatlymphocyteandmyeloidprogenitorsemergeasearlyasE7.
5withinthepara-aorticsplanchnopleura(pSp)[6]andthatexplantsofpSpdemonstratelong-termhematopoieticreconstitutionabilitywhereasyolksacexplantsdemon-strateonlyshorttermmyeloidreconstitutionability[7].
SimilarresultswerefoundinXenopusstudiesthatdemonstratedHSCemergencefromdorsalaortictissuethatderivedfromablastomereindependentoftheyolksac[95].
Theimplicationofthisisthatlong-termrepopulatingstemcells,orHSC,areintra-embryonicinorigin.
Workbyseveralgroupsledtothediscoveryofintra-aortichematopoieticclusters(IAHC)arisingfromtheendothelialliningofventralwallofthedorsalaortapriortotheirreleaseintocirculationandeventuallong-termcolonizationoffetalliverandmarrow,thuscorroboratingtheintra-embryonicoriginofHSC[43,53,55,57,96–101].
IAHChavebeenshowntoappeararoundE9.
5intheAGM,andpeakemergenceofHSCfromthemoccursbetweenE10.
5andE11.
5[21–23,96].
ThecellularcompositionandfunctionofIAHCbeforeandduringHSCdetectionhasbeeninvestigated,althoughtodateauniedcellsurfacemarkerproleofHSCgen-eratedwithintheembryohasnotbeenidentied,presentinganimportantroadblocktofurtheringourunderstandingofEHT.
IAHCatE10.
0werefoundtobephenotypicallyheterogeneousandcontainveryfewpro-genitors(averageof22),butnotablycontainpre-HSC(averageof12)capableoflong-termmultilineagehematopoieticreconstitutionpost-transplantation[102].
Acollectionofhematopoietic,stem,andendothelialmarkerssuchasc-Kit,CD31,CD34,SCA-1,MAC1,VE-cadherin,Tie2,Flk-1andCD45havebeenshowntobeexpressedbyHSCwithintheembryo,butthesearealsofoundonothercelltypes[47,57,99,103–106].
Intra-aortichematopoieticclustershavealsobeenshowntobecomposedofpre-HSCthatdifferentiallyexpressendothelialandhematopoieticsurfacemarkersincludingVE-cadherin,c-Kit,Ly6a,CD41andCD45,suggestingthatapost-hemogenicendothelialcellintermediatemayexistalongthetransitionfromendothelialcelltoHSPCwithintheseintra-aorticclusters[53,102,106–108].
TheremainingcelltypeswithinIAHCareyettobeidentied,butarehypothesizedtobecom-prisedoftheseimmaturepre-HSCthatwillmaturetowardaHSCfateviaEHT.
OncethehematopoieticclusterphaseofEHTiscomplete,datafromchickstudiesshowthattheSpecicationandfunctionofhemogenicendotheliumduringembryogenesis1553123splanchnopleuralendotheliumintheaorticoorisreplacedbysomiticendothelialcells,inpartofferinganexplanationfortheverybrieftimeframeinwhichvascularhemato-poiesisoccursduringdevelopment[109],althoughthishasnotbeenshowninmousemodels[110].
ThedorsoventralpolarityofIAHCemergenceappearstobecriticaltogoverningtheirultimatehematopoieticfate.
Thishasbeenshowninthechick[111]andmouse[112]tobeguidedbylocalpresenceofmesenchymallyderivedpro-hematopoieticventralizing(VEGF,bFGF,TGF-b,BMP4)oranti-hematopoieticdorsalizingfactors(EGFandTGF-a),thedownstreameffectsofeachimpacttheexpressionofcriticalhematopoietictranscriptionfactorsthatareinvolvedinEHT[14,111,112].
Importantly,sub-aorticmesenchymehasnotbeenshowntobeadirectsourceofhematopoieticcells[45],thuscorroboratingtheimportanceoftheselocallyderivedfactorsindrivinghematopoieticspecicationofvascularendothelialcells.
HSChavebeenfoundtohaveparticularenrichmentinthemiddlethirdoftheE11.
0dorsalaorta,immediatelyadjacenttooriginofthevitellineartery,suggestingthatadisturbanceinowatthejunctionofthesetwovesselsmayinteractwithmicroenvironmentalsignalstodriveHSCemergence[113].
ThepresenceofhematopoieticmediatorssuchasRUNX1intheventralaorticmesenchyme[56,57],andupregulationofgenesinvolvedincelldeath,adhesion,migration,aswellasvasculardevelopmentandhemato-poiesis[113],coupledwithrestrictionofdenitiveHSClocalizationtotheventralaorta[99]andevidencedemonstratingthatpara/peri-aorticsupportingcellsalsopromotehematopoiesis[111,112,114–116],suggeststhatwithintheAGMtherelikelyexistsaspecializedhematopoieticmicroenvironmentorniche[117]poweredbymastertranscriptionalregulators,theirdownstreameffectors,andextrinsicmodiersofthesesignalingcas-cadessuchasuidshearstress[118,119]thatpromoteinitialHSCdevelopmentfromhemogenicendothelium—thesearereviewedbelow.
RUNX1RUNX1(AML1)isasequence-specicDNAbindingproteinthatispartofafamilyoftranscriptionfactorscalledcorebindingfactors(CBF),andanessentialmasterregulatorofEHTduringaortichematopoiesis[58,120–122].
ConsistentwitharoleinEHT,RUNX1deciencyinembryonicstemcellspreventstheformationofbloodcellsfromhemogenicendothelium[42].
Itisexpressedinallsitesofdenitivehematopoiesisintheembryoandpre-cedesemergenceofHSPC.
Interestingly,thisisconservedacrossallvertebratespeciesstudied[123].
RUNX1isexpressedbymesenchymalcellsinthedorsalaortaandAGM,placenta,andinhematopoieticcellclustersinventral-dorsalaortaandvitellineandumbilicalarteries[56,57,123],butitsrequirement,whilecriticalfornormaldenitivehematopoieticdevelopment[124–126],aspre-viouslyreviewed[14],istransientinembryonicdevelopment,andisnotrequiredduringprimitivehema-topoiesis[42,121]orafterEHT[58].
RUNX1expressionhasbeenshowntorepresstheendothelialprogram,whileactivatingthehematopoieticprogram,asevidencedbysequentialemergenceofhematopoieticmarkersCD41andlaterCD45[42,106,108],atthetimeoftransitionfromhemogenicendothelialcellstoahematopoieticcellfate[120,123].
DeletionofRunx1doesnotpreventemergenceoftheCD41markeronVE-cadCD45-cells,buthasbeenshowntopreventtransitionofthesecellstoCD41CD45[127].
ThispatternedtransitionfromendothelialcellphenotypetoahematopoieticphenotypeisinpartcontrolledbybindingofmultiproteincomplexescontainingGATA,Ets(PU.
1)[128],andSCLfamilyfac-torstoRunx1enhancersknowntobeinvolvedinHSCemergence[14,129,130]andisalsomarkedbylossofexpressionofgenesassociatedwitharterialidentity,suchasSox17andNotch1[131],furthersupportingtheideaoftemporaltranscriptionalregulationgoverningcellfateasacentraltenetofEHT.
ProspectiveisolationandtranscriptionalanalysisofmurinehemogenicendothelialcellsusingatransgenicRunx1enhancer-GFPreportermodelhasdemonstratedthatbeginningatE9.
5,hemogenicendothelialcellscommittohematopoieticlineageinanorderedfashionmarkedbyanearlygraduallossofendothelialpotentialandincreaseinhematopoieticpotential.
Thisoccurswhilethehemogenicendothelialcellsstillremainpartoftheendothelialwall[59].
ThedifferentstagesofthetransitionfromendothelialtohematopoieticcellhavealsobeenshowntobeguidedbythetranscriptionalactivityofRunx1'stwo[132]promoterregions:theP1(distal)andP2(proximal)Runx1promoterscontrolexpressionofRunx1c(distal)andRunx1b(proxi-mal)isoforms[133,134].
RUNX1bisexpressedatlowlevelsinhemogenicendotheliumuntilE10.
5[135,136],whereasRUNX1cexpressionisinitiatedwithinHSPCandlossofendothelialphenotype[133,134,136].
TheP1promoterelementassociatedwithRUNX1cexpressionismorecomplexthantheP2promoterandcontainsmanybindingsitesforhematopoietictranscriptionfactors[132].
RUNX1btranscriptomeanalysisrevealsthatitactivatesgenesassociatedwithcelladhesion,ECMremodeling,integrinsignaling,andendothelialcellmigrationattheonsetoftransitionfromhemogenicendothelialcellstoHSPC[133].
RUNX1downstreamtargetssuchasGFI1/GFI1Barealsothoughttopromotedown-regulationofendothelialproteins,therebypromotingamorphologicchangefromattenedendothelialcellstoroundcells[137].
LackofGFI1Binembryosresultsinfailureofreleaseof1554E.
Gritz,K.
K.
Hirschi123hematopoieticcellsfromtheextraembryonicyolksacvasculatureintocirculation[137].
GenerationofP1andP2RUNX1-EGFPzebrashreporterlinesdemonstratedery-thromyeloidprogenitoremergencefromtheposteriorbloodislandat18hpfintheP1lineanddenitiveHSCemergenceintheAGMregionat22hpfintheP2line,furtherunderliningtheroleofalternativeRunx1promoterusageincellfatedeterminationviagenerationofspatiallysegregatedyettemporallysuccessiveHSPCemergence[138].
OtherworkfurtherdemonstratedthattheRUNX1cisoformwasindeedonlyexpressedatthetimeofemer-genceofdenitiveHSC(E10.
5-11.
5)inthemouseAGMandhuman(embryoidbodyday12),butretroviralover-expressionofbothRUNX1isoformsinmousemarrowandliverHSPCdemonstratednofunctionaldifferencebetweeneitherisoforminfurthergenerationofHSC,andinducedquiescenceinmouseHSCinvitroandinvivo.
Thissug-gestsadevelopmentallyimportanttimeframefortheRUNX1cisoforminspecicationofHSPCfromhemo-genicendothelialcellsatthetimeofEHT[139].
OtherstudieshaveidentiedaroleforbloodowinducedshearstressasaregulatorofRunx1expressionbytheendothe-lium[118,119].
StudiesutilizingconditionaldeletionofRunx1,drivenbyVav1promoterexpression,apan-hematopoieticgene,demonstratethatitshematopoieticrequirementonlyextendsthroughthetransitionfromendotheliumtoHSPC[58].
DependenceonRUNX1forgenerationofery-thromyeloidprogenitorcellsbeginsatE7.
5[140]andendsaroundE10.
5[141].
FurtherstudyofrequirementforRUNX1inhematopoieticdevelopmentutilizedtimedendothelial-specicdeletionofRUNX1activityshowedthatthetransitiontoRUNX1independentHSCformationoccursaroundE11.
5anddoesnotrequirefetallivercol-onization[141].
Tissue-specicrestorationofRUNX1functioninmurineTie-2-expressingcellsallowedforres-cueofembryonicdenitiveHSCpotential,allowingmutantstosurviveuntilbirthwithoutthepreviouslydescribedhemorrhagicphenotypeobservedinRunx1nullembryos.
TheseobservationssuggestthattheprimaryembryoniclethaldefectinRunx1mutantsisindeedhematovascularinnature[142].
Inadults,deletionofRunx1doesnotcauseareductioninHSPCnumbersinthemarrow;infact,itcausesanexpansionofthesecelltypes[123]andleadstohematologicabnormalities,suchasincreasedmyeloproliferationwithneutrophilia,thrombo-cytopenia,andincreasedextramedullaryhematopoiesis[143].
IntheabsenceofRUNX1duringdevelopment,primitiveerythropoiesisproceedsnormally,hemogenicendotheliumdevelops,butneithergenerationofmyeloidandlymphoidprogenitors,norHSCgenerationoccurs(reviewedin[14]),leadingtoanabsenceofIAHCandfetalliverHSC[54,56,57,144],andultimatelydeathbyE12.
5[144].
Interestingly,studiesofhematopoietictissuesfromRunx1haploinsufcientembryosyieldsvariabledegreesofHSCgenerationdependingonthetissuetypeisolated,suchthatculturedmutantAGMexplantsyieldfewerHSCthanwild-type,andyolksacandplacentaltissuesyieldincreasednumbersofHSC[145].
Ontheotherhand,AGM-derivedHSCfromRunx1haploinsufcientmicethataredirectlytransplantedintoirradiatedrecipientswithoutanintermediateculturestepdemonstratesincreasedgen-erationofHSC[57].
Collectively,thisbodyofevidencestronglysupportsaroleforRUNX1asacriticalregulatorofEHT,andthatmodicationofitsexpressionviainter-actionwithassociatingproteinswithinspecictissuemicroenvironmentsanddevelopmentaltimepoints,aswellasunderdifferentphysiologicconditions,impactsregula-tionofthehematopoietictransition[14,124,145].
SCL/LMO/GATATheregulatoryregionsofhematopoieticgenesareknowntobeboundbyRUNX1,asdiscussed,aswellasatran-scriptionfactorcomplexcomposedofstemcellleukemiaprotein(SCL)/Tcellacutelymphocyticleukemiaprotein-1(TAL-1),LIMdomainonly2(LMO2),GATA1,andGATA2([1,140,146–148],allofwhichhavebeenimplicatedinEHT.
LMO2isthoughttoactasabridgebetweenSCL/TAL-1andGATA-bindingproteinsinatranscriptionalactivatingcomplexthatdrivesvertebratehematopoieticspecication(reviewedin[1]).
StudiesofScl/Tal-1,Lmo2,andGata1/2mutantmouseembryosdemonstratethatdeletionofeachgeneresultsinmidgestationlethalityandimpaireddenitivehematopoi-esis[149–152].
SCLisahelix-loop-helixtranscriptionfactorthatfunctionsupstreamofRUNX1,andhasbeenshowntobeacriticalregulatorofhematopoiesisviagen-erationofTie-2hic-KitCD41-hemogenicendothelium[42].
SCLloss-of-functionstudiesshowthathematopoiesisisimpairedatboththelevelofstemcellformationandsubsequentdifferentiation[153].
Interestingly,multipleisoformsofSCL[SCLA(fulllength)andSCLB(N-ter-minaltruncated)]havebeenidentied[153],andmayplaydifferentialrolesindenitivehematopoiesis.
InvivotimelapseimagingstudiesdemonstratedselectiveSCLBiso-formexpressioninthedorsalaortaofhemogenicendotheliumjustpriortoEHT,andthisisoformmayactupstreamofRUNX1tomediateEHT.
SCLA,ontheotherhand,isexpressedinHSCaftertheEHTprocess,andiscriticalformaintenanceofthesenewlyborncellswithintheAGM[153].
GATA2isexpressedbythepara-aorticsplanchnopleuraandAGMregionsofthemouseembryo[154].
IthasbeenshowntohavearoleinproductionandexpansionofHSCSpecicationandfunctionofhemogenicendotheliumduringembryogenesis1555123intheAGMduringembryogenesisaswellasnormalexpansionoftheadultHSCpoolwithinthebonemarrow[155].
Gata2decientmicedieatE10.
5duetodefectiveprimitiveerythropoiesisandlackofHSPCgeneration[151].
ConditionaldeletionofaGata2cisregulatoryele-mentinmouseAGMresultsinreducedexpressionofhematopoietictranscriptionfactorsSCLandRUNX1,andhemogenicendothelialcellsofthemutantsfailtogenerateHSPC,leadingtoembryoniclethalitybyE13-14[156].
Additionally,VE-cadherin-drivendeletionofGata2pre-ventsgenerationofintra-aorticclustersinmousedorsalaortaandresultsinadeciencyoflong-termrepopulatingHSC[157].
Embryonicstemcellsderivedfromadultchi-mericmicedecientinLMO2alsofailtocontributetotheendotheliumoflargevessels[158,159]andbloodcellproduction[158,159].
MorpholinoknockdownofeitherScl[147]orLmo2[160]inzebrashresultsinsimilarlyimpairedexpressionofhematopoieticgenesandlossofIAHC.
Collectively,thesendingsimplicatethisgroupoftranscriptionfactorsashavingessentialrolesinEHTanddenitivehematopoiesis.
NotchInadditiontothepreviouslydiscussedroleforNotchsignalinginhemogenicspecication,NotchhasalsobeenshowninanumberofvertebratestudiestobeessentialfordenitiveHSCemergenceintheembryo[87,161,162].
ItisthoughtthatNotchsignalingmediatesHSCdevelopmentviaEHTbyinteractionsbetweenemergingHSPCexpressingNotchreceptorsandunderlyingendothelialcellsandstromaexpressingNotchligands[163,164].
Notch1andNotch4receptorsareexpressedspecicallyintheendothelialcelllayerofthedorsalaorta,andNotchsignalingpathwaycomponentssuchasDLL4,Jag1,Jag2,HES1,HRT1,HRT2,andGATA2areexpressedinboththeendothelialcelllayerofthedorsalaortaanditsemerginghematopoieticclustersbeginningbetweenE9.
5andE10.
5[165–167].
MouseandzebrashembryosthatlackNotchsignalingcomponentsdemonstratedefectiveorabsentdenitiveintra-embryonichematopoiesisandinabilitytoestablishpermanentself-renewingHSC(re-viewedin[1,165]).
In-vitroblockageofNotchsignalingusingDAPThasbeenshowntopreventEHTofculturedmurineE9.
5pSP/AGMcells[163].
ActivationofNotchsignalinginamodiedculturesystemofE11.
0pSp/AGMhemogenicendothelialcellsonimmobilizedchimerichumanDLL1resultsinemergenceofmultilineagehematopoieticprogenitorformationwithlong-termengraftmentcapability[163],furthersupportingthecriticalroleofNotchindrivingearlyhematopoiesis.
HSCgenerationinthehematopoieticclustersofthemurineAGMhasbeenshowntomostlydependonNotchligandJag1anditsregulationofGata2expression[167].
TheGata2promoterregioncontainstwoRBPJbindingsitesinvolvedinNotch-mediatedactivation[168]andnotsurprisingly,deletionofNotchtranscriptionalco-activatorRBPJleadstolossofHSPC,aswellaslossofGATA2expression,whichisnecessaryforcontinueddifferentiationofhematopoieticcells[166,169].
Notch-dependentGATA2bexpressionhasbeendemonstratedtoberequiredforRUNX1expressionwithinzebrashdorsalaortafromaveryearlydevelopmentalstage,anditsexpressionislim-itedtoemergingHSC[170].
EmbryosdecientforNotch1downstreamtargetsHES1andHES5havebeenshowntohaveintactarterialspecicationbuthavenotablehe-matopoieticdefectsmarkedbyabnormalproliferationofnonfunctionalHSPCandincreasedexpressionofhematopoieticregulatorsincludingGATA2,RUNX1,andc-Myb[168].
FurtherinterrogationoftheGata2promoterregioninthisstudyrevealedthatinadditiontocontainingRBPJbindingsitesessentialforinitiationofhematopoiesis,italsocontainedHESbindingsitesnecessaryforGata2downregulationinvolvedindrivingfunctionalhematopoi-esis[168].
ThishighlightsthecomplexityofNotchsignalinginthiscontext,asbothdrivingexpressionofandconcomitantlynegativelyregulatingakeyhematopoieticmediator.
PreviousworkshowingthatdifferentNotchligandsconferdifferentsignalstrengths[171,172]hasformedthebasisforrecentworksuggestingthatdeterminationofendothelialversushematopoieticfateinthedevelopingAGMismediatedbyligand-specicdifferencesinNotch1signalstrength.
UseofhighandlowsensitivityNotch1activationtrapmousemodelsdemonstratedthathematopoieticspecicationandrepressionoftheendothe-lialprogramisdrivenbylowlevelsofJag1mediatedNotchsignaling.
IntheabsenceofJag1,DLL4mediatedhighlevelsofNotchactivityinsteaddrivestheendothelial/ar-terialprogram[173].
TheauthorsofthisstudyproposeamodelwhereinJag1antagonizesDLL4-mediatedhighNotchsignalinginasubsetofendothelialcellstodrivethemtowardahematopoieticfateratherthananendothelialfate[173].
ThisfurtherhighlightsthecriticalroleofJag1,aspreviouslydiscussed,indrivingEHTandimportantlyalsoaddstothebodyofevidencethatendothelialandhematopoieticcellsrepresentdistinctlineages.
Inhumanembryonicstemcells,NotchactivationhasalsobeendemonstratedtobenecessaryforgenerationofCD45cells,andNotchsignalingviaHES1isnecessaryfortheirhematopoieticdifferentiation[174].
OtherstudiesinhumanembryonicstemcellsshowedthatligandDLL4isinducedviaNotchsignalinginasmallsubsetofendothelialprogenitorsduringhematopoieticdifferentiation,andthelevelofDLL4expressionappearstocorrelatewithhematopoieticversusendothelialfate.
Thatis,DLL4-high1556E.
Gritz,K.
K.
Hirschi123progenitorsareenrichedforendothelialpotential,whereasDLL4low/-coincideswithacquisitionofhematopoieticpotential[174].
Thus,amodelhasbeenproposed,whereinhemogenicendothelialcellsareDLL4low/-andareNotchactivatedvianeighboringDLL-highcellstopromotetransitionfromanendothelialstatetoCD45hematopoi-eticcellsthatclusterinafashionsimilartointra-aortichematopoieticclustersintheAGM[174].
RelatedstudiesalsodemonstratedafunctionalroleforDLL4inpromotingspecicationofhematopoieticcellsfromhematoendothe-lialprogenitors,whereinexpressionofDLL4increasednumbersofclonogenichematopoieticprogenitors,andinterestinglyskewedtheirfatetowardanerythroidlineage[174].
NocorrelationbetweenlevelsofJag1orJag2expressionwasnotedwithrespecttogenerationofhema-toendothelialprogenitorsorhematopoieticprecursorstherefrom,ashasbeennotedinothervertebratestudies,sug-gestingthathumanHSCdifferentiationmaybeaffectedbyotherNotchligands[174].
ArecentstudyinvestigatedNotch-regulatedelementsinvolvedincontrolofHSCgenerationinzebrash,mouse,andhumanembryonictissues.
ChIPonChIPanalysisagainstNotchco-activatorRBPJwasperformedandidentiedcandidatepromoterregionsofgenesregulatedbyNotchinhemaotpoietictissues.
ThemostsignicantresultwasenrichmentofCdca7,whichisshowntorecruitRBPJ,aswellasNotch1ICD.
CDCA7expressionwasshowntobeupregulatedinthehemogenicpopulationderivedfromhumanembryonicstemcellsinaNotch-dependentmanner.
Down-regulationofCdca7mRNAwasshowntoinducehematopoieticdifferentiationandconcomitantlydecreaseHSPCnumbers,suggestingthatitisaNotch-mediatedtargetinvolvedinemergenceofHSPC,butnotdifferenti-ationofthesecells[175].
Wnt/b-cateninThereisevidencethatNotch,Wnt,andBMPpathwaysinteracttogenerateHSCinthezebrashembryoandalsoareinvolvedindrivinghematopoieticdevelopmentfromembryonicstemcells[176,177].
Wntisawell-describedevolutionarilyconservedsignalingpathwayknowntomodulatemanyareasofembryonicdevelopment.
TheWntligandsareafamilyof19glycosylatedproteinsthatbindFrizzledreceptorsandLDLreceptorproteinco-receptorstotriggeravarietyofdownstreamresponsesincludingactivationofb-catenin(canonicalpathway)and/orJNKandPKC(noncanonicalpathway)thatmodulategeneexpressionprogramscriticalfornormalembryonicdevel-opment(reviewedin[176]).
EarlyevidencethatWnt/b-cateninhasaroleinembryonichematopoiesiscamefromzebrashstudiesshowingProstaglandinE2mediatesHSCformationintheAGMviab-catenin[177].
Recently,ithasbeenshownthatEMPcellsemergingfromvascularendotheliumintheyolksacdosoindependentofvascularidentityortheinuenceofcirculation,buttheiremergencerequiresintactWntsignaling[12].
IthasbeenshownthatWnt/b-cateninactivityistransientlyrequiredintheAGMofmouseembryosforemergenceandgenerationoflong-termHSC,aswellasproductionofhematopoieticcellsinvitrofromAGMendothelialprecursors[176].
Hemo-genicendothelialcellsfromAGMatE11.
5havealsobeenshowntohaveahighdegreeofactiveWntsignaling,asevidencedbythepresenceofhighlevelsofb-cateninincellsliningthedorsalaorta,closetoemerginghematopoieticclusters,butnotincellsbuddingoffofendotheliumorwithincirculatingplasma[75].
InhumanpluripotentstemcellmodelsofEHT,hemogenicendotheliumundergoesEHTtowardeitheraprimitiveordenitivehematopoieticfatedependingonpresenceofeitherinactive(primitive)oractive(denitive)Wntsig-naling[178].
Thisbodyofevidencesuggestsacontext-specicroleforWntsuchthatitisrequiredforthegen-erationofhemogenicendotheliumandlikelyinitiationofEHTbutgraduallybecomesdownregulatedasHSPCaregenerated[75].
HOX/SOXHomeoboxgenes,suchasHoxandSoxgenes,playakeyroleindeterminingcellidentityduringembryonicdevel-opment[179–182].
Hoxparaloguegroup3(HOXA3),specically,hasanimportantroleinendothelialandcar-diovasculardevelopment(reviewedin[183]).
Itsexpressionwasfoundtobehighinembryonicmesenchymaltissue,intermediateinthedorsalaortaandabsentfromtheyolksacatE8.
25andE8.
5whendenitivehematopoiesisisoccur-ringinthattissue.
Thisisconsistentwiththeideathatitpreventshematopoieticdifferentiationofendothelialpro-genitorsviadown-regulationofRUNX1,GATA1,GFI1b,IkarosandPU.
1.
Conversely,downregulationofHOXA3isconcomitantwithincreasedRUNX1expressionwithintheaorticendothelium,anditsre-expressioninembryonicstemcells,aswellasculturedmouseembryonictissues,causesretentionofanendothelialphenotype.
ThesedatasuggestanimportantroleforHOXA3inmaintenanceofanendothelialstatepriortohematopoietictransition[183].
EctopicexpressionofanotherHoxparalogue,HOXB4,inyolksacderivedhematopoieticprogenitorcellshasbeenshowntodriveconversiontoadenitiveHSCphenotypewithlong-termengraftmentpotential[184].
Additionally,severaltranscriptionalregulatorsofthedenitivehematopoieticprogramsuchasRUNX1,SCL/TAL1,GATA2,andGFI1havebeenshowntobedirecttargetsofHOXB4inembryonicstemcell-derivedhematopoieticprogenitors[185].
Specicationandfunctionofhemogenicendotheliumduringembryogenesis1557123SOX17isatranscriptionfactorexpressedinAGMhemogenicendotheliumdownstreamofHOXA3[183]atE8.
5–8.
75,andinadditiontoitsroleasacriticalfactorgoverningarterialidentity[186],ithasbeenshowntoberequiredforgenerationoffetalandneonatalHSC[187].
AlthoughSOX17isnotrequiredforthegenerationofadultHSC[120],itsoverexpressioninadulthematopoieticpro-genitorshasbeenshowntoconferfetalHSCcharacteristics[188].
Additionally,SOX17candriveexpansionofcellsthatexhibitdownregulationofendothelialmarkersandalsopossessanabilitytogeneratehematopoieticcells[120,187,189].
LossofSox17decreasesexpressionofNotch1inthemurineAGM,suggestingthatSOX17mediatesgen-erationofhemogenicendotheliumviaNotchsignalingduringdenitivehematopoiesis[187].
ConditionallossofSOX17mediatedrepressionofRunx1andGata2recentlyhasbeenshowntoresultinincreasedproductionofhematopoieticcellsfrommurineAGMendothelialcellsthatcanbedampenedbyanincreaseinNotchsignaling[131],suggestingthatfollowinghemogenicspecication,SOX17modulateshemogenicendotheliumbyactiverepressionofthehematopoieticprogrambytheSOX17/NotchaxisuntilinitiationofEHT.
Thishasbeencom-plementedbystudiesofmurineAGMhemogenicendothelialcellsundergoingEHTusingcombinedcorrelativescanningelectronmicroscopyandimmunou-orescencedemonstratinganincreaseinRUNX1levelsconcomitantwithdecreaseinnuclearSOX17,roundingofpreviouslyattenedAGMendothelialcells,andtheonsetofco-expressionofCD41andc-Kitonthesenewlyroun-dedcells[190].
SOX17hasalsorecentlybeenshowntobenecessaryforcommitmenttothedenitiveerythroidlin-eage[191].
OthercloselyrelatedSOXfamilymembers,SOX7andSOX18,havearoleinprimitivehematopoiesis.
SOX7andSOX18havetransientexpressioninhematopoieticprecursorsattheonsetofbloodspecicationandexpressionoftheseinearlyhematopoieticprecursorsfrommouseembryonicstemcellsandembryosenhancestheirproliferationwhileblockingtheirmaturation[189,192,193].
cMybThetranscriptionfactorcMybwasoriginallyidentiedasanuclearprotooncogeneinvolvedincertainaviancancersbutisalsoexpressedinHSPCpriortotheirdifferentiation.
MultiplemousestudiesutilizingcMybmutantsdemonstratethekeyroleplayedbycMybwithinavarietyofhematopoieticpathwayssuchaslymphoiddevelopment,HSPCmaintenance,andHSPCdifferentiation[194].
DeletionofcMybresultsinmurineembryoniclethalityatE15.
5duetolackoffetalliverhematopoiesis,specicallyimpairederythroidandmyeloiddevelopment[195].
AGMexplantsfromcMyb–/–mutantmicealsoyield100-foldfewerHSPC[196].
Lossoffunctionstudiesinzebrashhaveshownsimilarlyimpaireddenitivehematopoiesis,eveninthesettingofotherwisenormalprimitivehemato-poiesis[197,198].
ZebrashstudieshavealsodemonstratedaccumulationofHSPCwithintheventralwallofthedorsalaorta,theirsiteoforigination,andthatthisfailureofmigrationtoahematopoieticnichesuchasthezebrashcaudalhematopoietictissueandkidneymayinpartunderliethepreviouslydemonstratedfailureofdenitivehemato-poiesisincMybmutants[194].
OthermediatorsofHSPCgenerationfromhemogenicendotheliumG-proteincoupledreceptorsToidentifycandidateregulatorsofEHTonagloballevel,wholegenometranscriptomeanalysisofmurineaorticendothelialcells,hemogenicendothelialcellsandHSPCpopulationshasidentied530differentiallyexpressedgenesduringEHTwithparticularupregulationofthegeneencodingGproteincoupledreceptor56(Gpr56)andheptadtranscriptionfactorsSCL,LYL1,LMO2,GATA2,RUNX1,ERG,andFLI-1)[199,200].
TheheptadfactorswerebeenshowntobindtoGpr56enhancerregionsandregulateitsexpression,andknockdownofGpr56inzeb-rashresultedinhematopoieticdefectsthatcouldberescuedwitheithermouseorzebrashGpr56RNA,thusestablishingGPR56asanovelregulatorofEHTviaayettobeelucidatedmechanism[199].
AnotherGproteincoupledreceptor,GPR183,hasalsorecentlybeenshowntodampenNotchsignalinginzebrashmodelsviaitsrecruitmentofb-Arrestin-1andE3ligaseNEDD4,bothofwhichdegradeNotch1inhemogenicendothelialcellstopromoteEHT[201].
PurinesignalingPurines(suchasadenosine,ADP,andATP)exhibitextracellularsignalingactivitythatregulatesdiversephysiologicanddevelopmentalcellularfunctionsincludingautoregulationofbloodow,cellproliferationanddiffer-entiation,andstemcellregeneration,allviacellsurfacereceptors[202].
AdenosinesignalingatthelevelofthevascularendotheliumhasrecentlybeendemonstratedtoplayaroleinregulationofHSPCdevelopmentinbothzebrashandmice.
Inarecentstudy,elevatedadenosinelevelsinzebrashembryoswereshownincreasenumbersofRUNX1/cMybHSPCinthedorsalaortaviaincreasedexpressionofCXCL8(IL-8),whichhasbeenshowntotriggerproliferationofhematopoieticprogenitor1558E.
Gritz,K.
K.
Hirschi123cells[203].
Furthermore,inthesamestudy,alterationofadenosinesignalingviaknockdownoftheA2badenosinereceptorresultedindisruptionofboththegenerationofSCLhemogenicvascularendotheliumandtheendothe-lialtohematopoietictransition.
Theseresultsweremirroredinmouseembryonicstemcellcolonyformingassays,aswellasE10.
5AGMexplants,thatdemonstratedanincreaseinproductionofmultipotentprogenitorcolo-niesinthesettingofincreasedadenosinesignaling[202].
ChromatinremodelingTheroleofreprogrammingoftranscriptionalstatesinpromotinglineagecommitmentinHSCcontinuestobeofgreatinterestintheeld.
Theroleofchromatinmodi-cationasanimportantmediatoroftranscriptionalregulationofEHThasbeeninvestigated.
MajumderandcoworkersdemonstratedthatthehistonechaperoneHIRA(histonecellcycleregulation-defectivehomologA)regu-latesRUNX1activityduringEHT.
TheyshowedthatHIRAmediatedhistoneacetylationofRunx1activatesthedownstreamhematopoietictargetsofRUNX1andthatinHIRAdecientmutants,therewasnotonlyreducedexpressionofRunx1,butalsolackofchromatinreorgani-zationnecessaryforappropriateRUNX1bindingandtranscriptionalactivitytodrivehematopoiesis[204].
ChromodomainhelicaseDNA-bindingprotein1(CHD1),anATP-dependentchromatin-remodelingenzymewhoseactivationisassociatedwithanincreasedleveloftotalactivetranscriptioninmouseembryonicstemcells[205,206]hasalsorecentlybeenshowntobeessentialforsuccessfulEHT.
Endothelial-specicChd1mutants,whileabletoproduceintactE10.
5IAHC,thatshowintermediatemarkersofdifferentiationsuchasRUNX1andc-Kit,donotsuccessfullyinitiatedenitivehematopoiesisanddiebyE15.
5duetosevereanemiaandabsolutefailureofery-thropoiesis[207].
AnalysisofcellsfromthemutantIAHCdemonstratedthatthecellsintheclustersunderwentapoptosisratherthanhematopoieticspecication,asevi-dencedbyfailureofexpressionofmarkersofhematopoieticlineagesuchasCD45,andavefoldreductioninmyeloidcolonyformingpotential.
GlobalgeneexpressionanalysisofChd1mutantendotheliumatE10.
5inthisstudywasgenerallyfoundtobeunchangedwhencomparedtowild-typeendothelialcontrols,butnotablylackedactivationofasetof156genesimplicatedinhematopoiesisandgrowth.
Furthermore,hematopoieticprogenitorsunderwentaCHD-1dependentelevationinglobaltranscriptionalatthetimeofEHTthatwasneces-saryforinitialexpansionandsurvivalofHSPCbutnotfordifferentiationandmaintenanceofthedifferentiatedHSCpool[207].
CommittedhematopoieticprogenitorswithaVav-CreChd-1mutationdemonstratedperipheralbloodcompositioncomparabletowildtypemiceatoneyearofage.
Takentogether,theseresultssuggestapointedcriticalroleforCHD-1mediatedincreasedtranscriptionaloutputinordertosuccessfullycompleteEHTandestablishfunctionalhematopoiesis,butthatitisnotrequiredbeyondthepointofhematopoieticspecication[207].
HypoxiaandinammationTheintrauterineenvironmentisnotablyhypoxicandviatheactionofhypoxiainduciblefactortranscriptionfactors(HIF),transcriptionofgenesnecessaryforpromotingnormaldevelopmentinthisenvironmentisactivated(re-viewedin[208]).
ThemidgestationAGMandplacentahavebeenshowntoexhibitlocalizedareasofhypoxiaincludinghematopoieticclustersandsomeunderlyingendothelialcells.
TheroleofHIF1ainregulationofembryonichematopoiesiswasrecentlyshowntobenec-essaryforgenerationofHSPCfromtheAGMfromE9.
0toE11[208].
Recently,aninterestinglinkbetweenToll-likereceptor4(TLR4)inammatorysignaling,acascadepreviouslyassociatedwithstress-inducedhematopoiesisinadultmarrow,andembryonichematopoiesishasalsobeenestablished.
TLR4-NF-jBsignalingwasfoundtoregulatehemogenicendothelium-derivedHSPCdevelopmentinbothmiceandzebrashembryosviaactivationofNotchsignaling[209].
Thiscomplementsotherrecentstudiesinvestigatingtheroleofnon-infectious/sterilepro-inam-matorysignalingviaTNFaorinterferoninHSPCdevelopmentinzebrashandmice[210–212],suggestingthatalow-gradepro-inammatorystateisnecessaryfornormalHSPCemergenceevenunderlow-stressconditionsandmayinfactprimethehematopoieticsystemforreactivehematopoiesisinthesettingofexogenousstressorssuchasinfection[209].
SummaryandconclusionsInsummary,thedevelopmentalhierarchygoverninghematovasculardevelopmentiscomplexandmultifactorialandourunderstandingofitisstillinitsinfancy.
Overtimethestudyofhematologicdevelopmenthasfoundedmanyconceptscentraltotheeldofstemcellresearchasawhole[213].
Hematopoieticdevelopmentbeginsearlyinthemammalianembryointheextra-embryonicyolksacandplacentaandpeaksintheAGMregionbyE10.
5.
Itischaracterizedbyoverlappingwavesofgenerationofprimitivebloodcells,multilineagehematopoieticprogeni-torcells,andnallydenitivelong-termrepopulatingHSCviaahemogenicendothelialcellintermediate.
EmergenceofbloodcellsfromhemogenicendotheliumisknownasSpecicationandfunctionofhemogenicendotheliumduringembryogenesis1559123theendothelialtohematopoietictransition,whichisdrivenbyanincrediblycomplexandyettobefullyelucidatedinterplaybetweendevelopmentaltimingandtissuemicroenvironmentalfactors.
Thisinterplayisthoughttogovernatimedgraduallossofendothelialcharacteristics[105],accompaniedbychangesinlevelandtypeofexpressionofhematopoieticregulatorssuchasRUNX1[56,99,142]beginningintheextraembryonicyolksac,placentaandumbilicalvessels,aswellasthemidgestationaorta,andpossiblybeingcompletedinhematopoieticnichessuchasthefetalliver,andbonemarrow[18,19,99,102,105,106].
Still,thereremainanumberofunansweredquestionssurroundingtheprogressionfromembryonicmesodermtoendotheliumtoHSPCandadultHSC.
Ofparticularinter-est,asalreadymentioned,isstudyofthetissuespecicmicroenvironmentsinwhichtheseprogenitorcellsaregeneratedandmatureduringdevelopment,andtherolethatmicroenvironmentplaysindrivingtheformationofmul-tilineageHSPC[29].
Todate,thereislittleevidencetosupporttheexistenceofhemogenicendotheliuminlaterstagesofembryonicdevelopmentorpostnatally,buthumancordbloodhematopoieticprogenitorshavebeenshowntodifferentiateintoendothelialprecursorscapableofgeneratingfunctionalvasculatureinvivoandiffurtherinstructedbyhematopoieticgrowthfactors,rstswitchtotransitionalCD144CD45cellsandthentohematopoieticcellsperhapsduetotheinuenceoftheirtissuemicroenvironment[214].
Thisndingisofgreatinterest,asitsuggeststhathemogenicendotheliumisnotastransientinnatureaspreviouslythoughtandmayexistbeyondtheembryonicperiodasanuntappedreservoirofhematopoieticpotentialfortherapeuticapplications[214].
Efcientgenerationanddifferentiationofhematopoieticcellsinvitrotodatehasbeenfraughtwithtechnicaldif-cultyandthisdifcultyisalmostcertainlyexacerbatedbyaneedforamorecompleteunderstandingofinvivoendothelialandhematopoieticdevelopment(reviewedin[215]).
Furtherstudyoftheseprocesses,fromspecicationofhemogenicendotheliumtogenerationofHSPCviaEHT,andestablishmentoffunctionaldenitivehematopoiesisbothintheprenatalandpostnatalperiod,willfurtherourunderstandingofdevelopmentalhematopoiesis,aswellasadulthematopoiesisinbothhealthanddisease.
Itwillalsohighlightthecellularandmolecularunderpinningsoftheevolutionofapopulationofstemandprogenitorcellsthatholdgreatpromiseforuseinclinicaltherapiesforhema-tologic,oncologic,vascular,andimmune-mediatedpathologies.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
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