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The Role of Estrogen Signaling in the Induction, Specification, and Proliferation of Hematopoietic Stem Cells

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The Role of Estrogen Signaling in the Induction, Specification, and Proliferation of Hematopoietic Stem Cells The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters. Citation Accessed Citable Link Terms of Use Carroll, Kelli Jane The Role of Estrogen Signaling in the Induction, Specification, and Proliferation of Hematopoietic Stem Cells. Doctoral dissertation, Harvard University. February 28, :10:14 AM EST This article was downloaded from Harvard University's DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at (Article begins on next page) TheRoleofEstrogenSignalingintheInduction,Specification,andProliferationof HematopoieticStemCells Adissertationpresented by KelliJaneCarroll to TheDivisionofMedicalSciences inpartialfulfillmentoftherequirements forthedegreeof DoctorofPhilosophy inthesubjectof DevelopmentalandRegenerativeBiology HarvardUniversity Cambridge,Massachusetts April2014 2014KelliJaneCarroll AllRightsReserved. DissertationAdvisor:ProfessorTristaNorth KelliJaneCarroll TheRoleofEstrogenSignalingintheInduction,Specification,andProliferationof HematopoieticStemCells Abstract Hematopoietic Stem Cells (HSCs) are characterized by their ability to both selfq renew and give rise to all lineages of the blood system. A recent chemical genetic screen identified17βqestradiol(estrogen)asanovelmodifieroftheexpressionoftheconserved HSC markers runx1 and cmyb in the AortaQGonadQMesonephrosofdevelopingzebrafish. Exposure to exogenous estrogen during the development of the hematopoietic niche impeded specification of hemogenic endothelium and the subsequent emergence of HSCs via antagonism of somiticqderived VEGF signaling. Conversely, inhibition of endogenous estrogen activity increased the number of functional HSCs present in the embryo and resulted in higher expression of VEGF target genes, suggesting that endogenous estrogen actstodefinetheventrallimitofvegfactivityandhemogenicendothelialspecification. In contrast, when embryos were exposed to estrogen after niche specification, markers of HSCs were increased, indicating that estrogen has a biphasic effect on HSC formation;thiseffectappearstobeatleastpartiallymediatedbyenhancedcellcyclingof thehscpopulation.estrogenexposureduringprimitiveerythropoiesislikewiseincreased the number of erythroid progenitors in the embryo, but their maturation into functional erythrocytes was impaired. Inhibition of erythrocyte maturation is also conserved in a mammalianmodelofin+uteroexcessestrogen,+causingpropensityforembryoniclethality.++ iii Treatment of adult zebrafish with exogenous estrogen after ablation of the hematopoietic system by irradiation revealed that elevated estrogen levels improved hematopoietic regeneration. Consistent with a role for hormonal regulation of HSC homeostasis, accelerated recovery of hematopoietic stem and progenitor numbers was observed in female fish compared to males, suggesting an endogenous difference in regenerative capacity between the sexes. Together, these data identify multiple distinct roles for estrogen in HSC biology and indicate it is a physiologically relevantregulator of HSCdevelopmentandhomeostasis. iv TableofContents Abstract.....iii CitationstoPublished/SubmittedWork......vi Acknowledgements.....vii Chapter1:Introduction..1 Chapter 2: Estrogen Defines the DorsalFVentral Limit of VEGF Regulation to Specify thelocationofthehemogenicendothelialniche...32 Introduction Results 37 Discussion.82 MaterialsandMethods.88 Chapter3:EstrogenEnhancestheNumberofHematopoieticProgenitorsduringboth DevelopmentandRegeneration 91 Introduction 93 Results 95 Discussion..110 MaterialsandMethods..113 Chapter4:Discussion.115 Appendix I:Cannabinoid ReceptorF2 Signaling Regulates Embryonic Hematopoietic StemCellFunctionviaProstaglandinE2andPFselectin. 130 AppendixII:SupplementalMethodsandReagents.174 References v CitationstoPublished/SubmittedWork PartsofChapter1areinpress: Carroll KJ and North TE. Oceans of Opportunity: Exploring Vertebrate HematopoiesisintheZebrafish.+(2014)+Experimental+Hematology.+In+Press. PartsofChapter2areinpress: CarrollKJ,EsainV,GarnaasMK,CortesM,DoveyMC,NissimS,FrechetteG,Cutting CC,KwanW,HarrisJM,LiuS,GorelickD,HalpernM,LawsonN,GoesslingW,North TE. Estrogen Defines the DorsalQVentral Limit of VEGF Regulation to Specify the LocationoftheHemogenicEndothelialNiche.(2014)Developmental+Cell.In+Press.+ + AppendixIisinrevision:+ + EsainV,CarrollKJ,CortesM,LiuS,KwanW,FrechetteGM,ShewardL,GoesslingW, North TE. Cannabinoid ReceptorQ2 Signaling Regulates Embryonic Hematopoietic StemCellFunctionviaProstaglandinE2andPQselectininZebrafish.(2014)Blood.++ In+Revision. vi Acknowledgements I+am+very+grateful+to+many+people+for+their+assistance+with+this+thesis.++ + First,+thank+you+to+Trista,+for+being+the+best+PI+I+could+have+asked+for+and+for+pushing+me+ farther+than+i+thought+possible.+five+years+ago,+i+would+not+have+dreamed+how+deeply+i+would+ fall+in+love+with+bench+science+and+the+passion+i+would+find+for+research.++your+support,+ guidance,+and+mentorship+were+invaluable+throughout+this+process.+thank+you.+ + Thank+you+to+the+members+of+my+Dissertation+Advisory+Committee+(Caroline+Burns,+Pat+ D Amore,+Hanno+Hock,+and+Scott+Armstrong)+for+your+helpful+comments+and+support.+ + To+the+members+of+the+North+Lab:+Thank+you+for+the+laughs,+for+answering+my+crazy+ questions,+and+generally+being+a+fun,+collaborative,+and+supportive+group+of+people.+each+of+ you+made+coming+into+lab+every+day+great+fun+and+never+a+burden.+ + Thank+you+to+Wolfram+and+the+other+members+of+the+Goessling+lab+for+suggestions+and+ technical+help.+ + Thank+you+to+my+BBS+classmates+for+the+fun+nights+out+and+the+weekly+lunches.+I m+always+ aware+of+how+lucky+i+am+to+be+surrounded+by+so+many+smart,+amazing+people.++ + To+my+nonSBBS+friends:+I+am+continually+thankful+to+have+so+many+wonderful+friends+both+in+ Boston+and+across+the+country.+Thank+you+for+reminding+me+that+there+is+a+world+beyond+the+ lab+and+for+expanding+my+horizons+by+inviting+me+into+your+lives.+ + I+am+forever+grateful+to+my+family+for+their+love+and+support.+Bruce,+Sarah,+and+Emma:+Thank+ you+for+the+phone+calls,+texts,+ s,+and+videos.+i+am+deeply+cognizant+of+how+lucky+i+am+to+ have+a+brother+and+sistersinslaw+who+are+not+just+family+but+friends+as+well.+and+thank+you+for+ letting+me+steal+your+child+everyone+once+in+awhile+for+some+fun being+an+aunt+is+a+total+ blast+and+i+thoroughly+enjoy+corrupting+emma+every+chance+i+get Finally,+thank+you+to+my+parents,+for+teaching+me+to+dream+big+while+never+losing+sight+of+who+ I+am.+Your+tireless+love,+advice,+and+support+have+enabled+me+to+follow+my+heart,+even+when+it+ led+me+far+from+home.+thank+you+for+showing+me+the+world+and+for+always+standing+behind+ me.+i+love+you+both.++ vii To+my+parents,+Dennis+and+Connie+Carroll,+ + for+your+endless+love+and+support.+ + Thank+you. viii Chapter1: Introduction 1 IdentificationandCharacterizationoftheHematopoieticStemCell Hematopoiesisistheprocessbywhichallthebloodcellsofanorganismareformed. The hematopoietic stem cell(hsc) sits atop the hierarchy of hematopoietic development andischaracterizedbyitsabilitytobothselfqrenew and give rise to all the different hematopoietic lineages for the lifetime of an organism(figure 1.1).(Cumano and Godin, 2007; Orkin and Zon, 2008). While the concept of a hematopoietic stem cell had been proposed as early as the late 1800s (RamalhoQSantos and Willenbring, 2007), the first formalexperimentalevidencethatsupportedtheexistenceofanhsciscreditedtotilland McCullouchwhoshowedthattransplantationofmurinebonemarrowcellsintoirradiated recipientsledtheformationofspleencoloniesatarateapproximatelyproportionaltothe number of transplanted cells (McCulloch and Till, 1960; Till and McCulloch, 1961). This discovery initiated intense research into the genetic and molecular mechanisms that regulatethedevelopmentandmaintenanceofthehematopoieticsystem,helpingtomakeit one of the most wellqcharacterized organs to date. The discovery of HSCs also revolutionized the treatment of many hematological malignancies including leukemia, lymphoma, and other hematopoietic disorders such as anemia through the use of bone marrow, and later, mobilized peripheral blood or cord blood transplantation therapy (Copelan,2006). VertebrateHematopoiesis WhilethelongQtermrepopulatingHSC(LTRQHSC)istheultimategeneratorofallthe lineagesinthebloodsystem,itisalargelyquiescentcellafteritisestablishedduring 2 Figure1.1 Thehematopoieticstemcell(HSC)isresponsibleforthegenerationofalllineagesof thebloodsystemincludinglymphoid,myeloid,anderythroidcells.reprintedby permissionfrommacmillanpublishersltd:oncogene;(larssonandkarlsson, 2005),copyright development(cheshieretal.,1999).instead,thebulkofthecelldivisionsandproliferation inthehematopoieticsystemareperformedbyshortqlivedmultipotentprogenitorscells, sometimes termed shortqterm HSCs (STQHSCs), and downstream committed precursor populations,such as Common Lymphoid and Myeloid Progenitor cells (Passegué et al., 2005).Theseprogenitorsgiverisetothetwomajorlineagesofthehematopoieticsystem, erythroqmyeloidandlymphoid(wangandwagers,2011).theprimaryfunctionofmyeloid cells, including macrophages, neutrophils, and granulocytes, which make up the innate immune system, is to fight against infection in the host animal, while lymphoid derived cells(tandbcells)composetheadaptiveimmunesystemandaredesignedtorecognize prior foreign pathogens to facilitate a quick biological response to secondary exposure (IwasakiandMedzhitov,2010;Tredeetal.,2004). Erythroid, or red blood cells, are hemoglobin carrying erythrocytes that are responsibleforthetransportofoxygenthroughouttheorganism;defectsinerythropoiesis give rise to many different forms of anemia (Dzierzak and Philipsen, 2013). The maturationalcascadeoferythrocytesisquitewellunderstoodandrequiresmanydifferent genesincludingscl+(shivdasanietal.,1995),lmo2+(warrenetal.,1994),gata2+(tsaietal., 1994)+and+Gata1+(Fujiwaraetal.,1996;Pevnyetal.,1995;1991);++lossofanyofthesegenes is embryonic lethal, underscoring their critical roles in early development.+ Gata1 is considered a master regulator of erythrocyte maturation as it is required to for the activationofmanyerythroidgenes(ferreiraetal.,2005;welchetal.,2004).thecombined transcriptionalactivationofthesegenes,alongwithothercoqfactors,ultimatelyinteractto formafunctionalhemoglobinpositive,oxygenqcarryingerythrocyte,whichisessentialfor embryonicdevelopment(mcgrathandpalis,2008). 4 Figure1.2(AdaptedfromTristaNorth). ThesitesofHSCdevelopmentchangethroughoutdevelopment.Acrossvertebrate species,hscsarebornintheagmandsubsequentlymigratetootherlocations beforereachingthebonemarrow(humanandmouse)orkidneymarrow (zebrafish),thesitesofhematopoiesisinadults. 5 TheDevelopmentalBiologyofMurineHSCs IncontrasttothehierarchicaldifferentiationfrommultipotentHSCsthatoccursin theadult,duringdevelopment,hematopoiesisinitiatesasaseriesofsequentialwaves,each with increasing competence (Figure 1.2). The first wave of hematopoiesis, termed primitive, gives rise to a transitory population of both erythroid and myeloid cells; these cellsarelargelyformedintheextraembryonicyolksacinmammalsstartingatembryonic day 7.5 (Dzierzak and Speck, 2008). During murine development, the first true hematopoietic stem cells emerge in the aortaqgonadqmesonephros (AGM) beginning at approximatelyembryonicday10.5(medvinskyanddzierzak,1996;medvinskyetal.,1993; Sanchezetal.,1996);thesecellsareresponsibleforrepopulatingthebloodsystemofthe animal for its entire lifetime, making this a critical step during early embryonic development(dzierzakandspeck,2008;medvinskyetal.,2011;orkinandzon,2008).a similar process occurs during human gestation from roughly days 27 to 40 of gestation (Tavian and Peault, 2005). Interestingly, while the AGM is the location of the initial formation of HSCs, it only remains a hematopoietic site for a short period of time during development. Instead, the HSCs from the AGM migrate to the fetal liver at ~E14.5 in the mouse,wheretheyarejoinedbyasecondwaveofde+novoproducedhscsderivedslightly later in the placenta, as well the yolk sac; together, these cells continue to proliferate as well as differentiate to meet the needs of the growing fetus (Ema and Nakauchi, 2000; MikkolaandOrkin,2006;Morrisonetal.,1995;Mülleretal.,1994).Finally,shortlyafter birth, HSCs egress from the fetal liver and travel to the bone marrow where they will remaininvaryingstatesofactivityandquiescenceforthelifetimeoftheorganism,asmany cell types in the bone marrow microenvironment (niche) work together to regulate HSC 6 maintenanceandhomeostasis(christensenetal.,2004;orkinandzon,2008;zanjanietal., 1993). Interestingly, neither the fetal liver nor the bone marrow is thought to be responsiblefortheproductionofde+novo+hscs,makingtheearlieststepsinhematopoietic developmentintheagmcriticalforthelongqtermviabilityoftheorganism(dzierzakand Speck,2008). HSCsArisefromHemogenicEndotheliumintheAGMofDevelopingVertebrates Ithasbeenrecognizedsinceatleastthe1920sthatcertainendothelialcellsinthe bloodvesselsofdevelopingembryosappeared hemogenic (Maximow,1924).However,it was unclear if HSCs were generated de+ novo in the AGM or if they arose as a result of migration from another spatial location in the developing embryo. Early work utilized chickqquail chimeric grafts to demonstrate that definitive hematopoietic stem cells arise fromanintraembryoniclocation,ratherthanfromtheextraembryonicyolksac,whichwas the accepted source of definitive HSCs at the time (DieterlenQLievre, 1975). The intraembryonicgenerationofhscswaslaterconfirmedusingexplantculturesofdissected AGMs.ThisworkrevealedthatHSCsintheAGMaregeneratedautonomously,asHSCswere formedintheabsenceofbothbloodflowandotherpotentialcellularsources(medvinsky anddzierzak,1996).morerecentworkhasfocusedonelucidatingthecellswithintheagm thatareresponsibleforthegenerationofhscs.therelatedontogenyofthefirstdefinitive HSCs and the vessels is supported by multiple pieces of evidence including the closely related spatial emergence of budding clusters of HSCs in the wall of aortic specified endothelium as well as a high degree of expression of common genetic markers and regulators(cumanoandgodin,2007). 7 Recent studies have largely indicated that HSCs arise from the endothelial cells liningtheventralwallofthedorsalaorta.theemergenceofhscsfromthewalloftheagm, thestartofaprocesswherebynewlyproducedhscsbudfromtheendothelium,enterinto thecirculationoftheembryoandtraveltoseedthenextsiteofhematopoiesis,wastermed endothelial to hematopoietic transition (EHT) for its similarities to extravasation in cancer (Kissa and Herbomel, 2010). EHT appears to be highly conserved across vertebrates, as similar movements were noted in both murine (Boisset et al., 2010) and zebrafishsystems(bertrandetal.,2010;kissaandherbomel,2010).thoughitisnowwellq establishedthathscsarebornfromhemogenicendothelialcellsinthewallofthedorsal aorta,thesignalsthatcontrolthespatiallocalizationofhscstotheventralwalloftheaorta arenotwellelucidated,norarethosethatinitiatetheonsetofthisprocess. Runx1isaCriticalRegulatorofHSCDevelopment While many genes play important roles in the development of the hematopoietic system, one of the best characterized is Runx1, also known as AML1/Cbfa2. Runx1 knockoutmicedieat~e12.5duetohemorrhaginginthecentralnervoussystem;theyalso displayed deficits in fetal liver hematopoiesis (Wang et al., 1996). Later studies revealed thatrunx1isexpressedinbothhscsandtheendothelialcellsfromwhichhscsemergeat E10.5 (Figure 1.3); Runx1 knockout mice were also shown to lack AGM HSCs at E10.5, suggesting that their fetal liver hematopoiesis defects result from a lack of definitive hematopoiesisintheagmratherthanafailureofthehscstohometothefetalliver(north etal.,1999).prospectiveisolationofrunx1positivecellswasalsoshowntoenrichforadult 8 Figure1.3(AdaptedfromTristaNorth). AtE10.5,Runx1(asindicatedbyblue)isexpressedinendothelialcellsintheAGM andumbilicalandvitellinearteriesaswellasinhscs repopulatingactivityaftertransplant,confirmingthattheexpressionofrunx1doesindeed markthefirstdefinitivehscs(northetal.,2002).morerecentworkhasdemonstratedthat Runx1 is required for the endothelial to hematopoietic transition that occurs during the buddingofhscsfromtheendothelium,butnotafter(chenetal.,2009),helpingtoprovide temporalinformationontheroleofrunx1inhematopoieticdevelopment.whilerunx1is largelyrecognizedforitsseminalroleinhscspecification,itisalsocommonlymutatedin hematological malignancies, suggesting that it continues to play a role in hematopoietic homeostasis,evenafterthedevelopmentofagmhscs(cohen,2009).consistentwiththis 9 idea,hscsintheadultexpressrunx1,asdolymphoidandmyeloidsubtypes,indicatingit remainsanimportantregulatorofhematopoiesis,althoughitspreciseroleintheadulthas notbeenfullycharacterized(lorsbachetal.,2004;northetal.,2004). ZebrafishasaModelSystemforDevelopmentalBiology Whilethezebrafish(Danio+rerio)wasfirstsuggestedforuseinhematologyresearch in1963(colleqvandevelde,1963),itisonlyinthelast~20yearsthatithastrulyrisento prominence as one of the preeminent systems for hematopoietic stem cell(hsc) biology, particularlyintheareasofdevelopmentandregeneration.thezebrafishhasemergedasa highly tractable model system for scientific research due in large part to the external fertilizationofembryosandtheiropticallycleardevelopment,allowingforrealqtimein+vivo observation of developmental processes. Additionally, the ability of fecund females to lay hundredsofembryosperweekenablesrapidhighqthroughputexperimentationandstrong statisticalanalysisofphenotypes.zebrafishareparticularlyusefulforhematologyresearch duetothehighconservationofgeneticfactorsregulatingblooddevelopmentaswellasthe structure and function of hematopoietic cell types, and the ability to visualize circulating erythrocyteswithonlyadissectingmicroscope. HematopoiesisisHighlyConservedintheZebrafishModel As in all other vertebrates analyzed to date, zebrafish hematopoiesis occurs in multiple phases (Figure 1.4 and Figure 1.5). Primitive hematopoiesis, the first wave, occursfrom~12to24hourspostfertilization(hpf)intwoanatomicallydistinctlocations: asectionofposteriorlateralmesodermcalledtheinnercellmassgivesriseprimarilyto 10 Figure1.4(AdaptedfromTristaNorthandDavidTraver). Primitive erythropoiesis in the zebrafish occurs in the Inner Cell Mass (ICM). The first definitive HSCs are born in the AortaQGonadQMesonephros (AGM) before migrating to the Caudal Hematopoietic Tissue (CHT), followed by the thymus and kidneymarrow,thesitesofhematopoiesisintheadult. + cellsoferythroidlineage(detrichetal.,1995),whiletherostralbloodislandintheanterior portion of the embryo gives rise to a primitive macrophage population (Herbomel et al., 1999;Lieschkeetal.,2002).Theprocessoferythropoiesisrequiresmanyofthesamegenes that are utilized during primitive hematopoiesis in other vertebrate species including scl+ (Dooley et al., 2005),gata1+ (Lyons et al., 2002),lmo2+ (Patterson et al., 2007),andtif1+ (Ransometal.,2004)+whilethegenerationofmyeloidcellsrequirespu.1andcebpα+(Hsu etal.,2004;lyonsetal.,2001;suetal.,2007).atransientwaveofdefinitivehematopoietic progenitors has also been recently identified, termed erythromyeloid precursors(emps), thatarepresentintheembryopriortotheemergenceoftruemultiqlineagehscs(bertrand et al., 2007a). These progenitors give rise to both definitive erythroid and myeloid (neutrophilic granulocytes, monocytes, and macrophage) colonies in culture. However, EMPswereneverobservedtopopulatethekidneymarroworthymusin+vivo,indicatinga 11 Figure1.5: Schematicrepresentationofembryonichematopoieticdevelopmentshowinglineage relationships between the hematoqvascular populations. Solid lines indicate known lineagechoiceswhiledottedlinesindicateputativedecisions. lackoflymphoidpotentialandsupportingth
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