Grigorieva&ThakkerTranscriptionfactorsinparathyroiddevelopment
Table2.Continued
Transcriptionfactor(TF)Six1
Siteofexpression
Positionintheparathyroidgeneticpathway
Phenotypeofnull(?/?)mouse
Mutations(human)
FunctionHomeodomainTF
Reference95,111
Downstreamof:Expressedinthe
Tbx1,Fgf8andpharyngeal
Wnt,Eya1endoderm,
Upstreamof:mesenchyme,and
[unknown]ectodermfrom
earlystages;itsexpressionintheendodermandectodermisEya1-dependent
Tbx1
Gcm2
Downstreamof:Expressedin
Shhpharyngeal
Upstreamof:endodermandthe
Gcm2mesodermalcore
ofthepharyngealarches(butnotintheneuralcrest-derivedmesenchymeofthepharyngealarches)
TFwithagcm-DNAExpressedatE9.5inDownstreamof:
Hoxa3,Pax1/9,bindingmotifthe3rdpouch
Eya1,Six1,Shhpharyngeal
Upstreamof:Pthendoderm,
speci?callyintheparathyroiddomainofparathyroid/
thymusprimordialT-boxTF
AbsenceofthymusandBORsyndromeby
disruptionofparathyroid.Although
EYA1–SIX1–DNAthepatterningofthe
complexes3rdpouchinto
thymus/parathyroidprimordiaisinitiated,theendodermalcellsofthethymus/
parathyroidrudimentsfailtomaintaintheexpressionoftheparathyroid-speci?cgeneGcm2andthethymus-speci?cgeneFoxn1
Tbx1–/–dieatbirthandDiGeorgesyndrome
type2(includingdisplayawiderangeof
hypopara-developmental
thyroidism)anomalies
encompassingalmostallofthecommonDGSfeatures,
includinghypoplasiaofthethymusandparathyroids
26,34
Gata3Dual-zinc?ngerTF
ExpressedinitiallyinDownstreamof:
[unknown]thethymus
Upstreamof:domainofthe
Gcm2commonparathy-roid/thymus
primordiumatE10.5,byE11.5expressionof
GATA3isswitchedtotheparathyroiddomain
Gcm2–/–areviableandIsolatedhypopara-11,54,58,114
thyroidismfertile.Absent
parathyroidglands.Micede?cientforGcm2revealedthatPthisnotexpressedintheparathyroidanlagealthoughparathyroidlikecellscharacterizedbyPax9expressionarestillpresentatE14.5
SmallsizeorabsenceofHypoparathyroidism39,48theparathyroidsinthedeafness,renal
+/?
Gata3dysplasia(HDR)and
syndromeGata3?/?embryos,
respectively,duetothemarkedlyreducednumberof
Gcm2-expressingcellsinthe3rdpharyngealpouch
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mutationsinvolvingGATA3ZnF2ortheadjacentbasicaminoacidswerefoundtoresultinalossofDNAbinding,whereasthoseinvolvingZnF1eitherleadtoalossofinteractionwithFOG2ZnFsoralteredDNA-bindingaf?nity.40,42,43These?ndingsareconsistentwiththeproposedthree-dimensionalmodelofGATA3ZnF1,whichhasseparateDNAandproteinbindingsurfaces.40,42,43Thus,theHDR-associatedGATA3mutationscanbesubdividedintotwobroadclasses,whichdependuponwhethertheydisruptZnF1orZnF2andtheirsubsequenteffectsoninteractionswithFOG2andalteredDNAbind-ing,respectively.Themajority(>75%)oftheseHDR-associatedmutationsarepredictedtoresultintruncatedformsoftheGATA3protein.Eachprobandandfamilywillgenerallyhaveitsownuniquemutation,andthereappearstobenocorre-lationwiththeunderlyinggeneticdefectandthephenotypicvariation—forexample,thepresenceorabsenceofrenaldysplasia.Over90%ofpa-tientswithtwoorthreeofthemajorclinicalfea-turesoftheHDRsyndrome—thatis,hypoparathy-roidism,deafness,orrenalabnormalities—haveaGATA3mutation.42Theremaining10%ofHDRofpatients,whodonothaveaGATA3muta-tionofthecodingregion,mayharbormutationsintheregulatorysequences?ankingtheGATA3gene,orelsetheymayrepresentheterogeneity.ThephenotypesofHDRpatientswithGATA3muta-tionsappeartobesimilartothosewithoutGATA3mutations.42
TheHDRphenotypeisconsistentwiththeex-pressionpatternofGATA3duringhumanandmouseembryogenesisinthedevelopingkidney,oticvesicle,andparathyroids.However,GATA3isalsoexpressedinthedevelopingcentralner-voussystem(CNS)andthehematopoieticorgansinmanandmice,andthissuggeststhatGATA3mayhaveamorecomplexrole.Indeed,studiesofGata3+/?andGata3?/?micehaverevealedim-portantrolesforGata3inthedevelopmentofthebrain,spinalcord,peripheralauditorysystem,Tcells,fetalliverhematopoiesis,andurogenitalsystem.45Gata3?/?micediebetweenE11.5andE12.5,45butGata3+/?miceareviable,appeartobenormalwithanormallifespan,andarefer-tile.45However,Gata3+/?micehavehearinglossandparathyroidabnormalities.Thishearinglossisassociatedwithcochlearabnormalities,whichcon-sistofasigni?cantprogressivemorphologicalde-generationthatstartswiththeouterhaircellsattheapexandeventuallyinvolvesalltheinnerhaircells,pillarcells,andnerve?bers.46,47Thesestud-ieshaveshownthathearinglossinGata3haploin-suf?cientmicecommencesintheearlypostnatalperiodandisprogressivethroughadulthood,andthatitisperipheralinoriginandispredominantlyduetomalfunctioningoftheouterhaircellsofthecochlea.46,47
Gata3lossalsoresultedinparathyroidabnormal-ities.Thus,Gata3?/?andGata3+/?embryoslackedorhadsmallerparathyroid-thymusprimordia,re-spectively,48andtheparathyroidsofadultGata3+/?micedidnotenlargeorhaveanincreasedprolifer-ationrateinresponsetohypocalcemia,whichwasinducedbyalow-calcium/vitaminDdiet.More-over,theadultGata3+/?micehadaninadequateincreaseinplasmaPTHinresponsetotheinducedhypocalcemia.These?ndingsintheGata3+/?miceareconsistentwiththeobservedhypocalcemiathatoccursinassociationwithinappropriatelynormal-orlow-plasmaPTHconcentrationsinpatientswhohaveHDRsyndromeduetoGATA3haploinsuf-?ciency.37,39ThesmallersizeorabsenceoftheparathyroidsintheGata3+/?andGata3?/?em-bryos,respectively,isassociatedwithamarkedlyre-ducednumberofGcm2-expressingcellsinthethirdpharyngealpouch,anditislikelythatGATA3iscrit-icaltomaintainingdifferentiationandsubsequentsurvivalofparathyroidandthymusprogenitorcells.Indeed,GCMB,whichisthehumanhomologueofmouseGcm2,hasbeenshowntobetranscription-allyregulatedbyGATA3.48
ExaminationofGata3?/?embryoshasrevealedavarietyofabnormalitiesthatincludedmassivein-ternalbleeding,resultinginanemia,markedgrowthretardation,severedeformitiesofthebrainandspinalcord,ahypopigmentedretina,grossaber-rationsinfetalliverhematopoiesis,atotalblockofTcelldifferentiation,andaretardedormiss-inglowerjawarea.45,49TheseGata3?/?micehadananatomicallynormalsympatheticnervoussys-tem,yetthesympatheticganglialackedtyrosinehy-droxylaseanddopaminebeta-hydroxylase,whicharekeyenzymesthatconverttyrosinetoL-DOPAanddopaminetonoradrenaline,respectively,inthecatecholaminesynthesispathway.Thus,theGata3?/?micelackednoradrenalineinthesympa-theticneurons,andthiswascontributingtotheearlyembryoniclethality.49Feedingofcatecholamine
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intermediatestothepregnantdams,helpedtopar-tiallyrescuetheGata3?/?embryosfromE12.5toE16.5.49Theseolder,pharmacologicallyrescuedGata3?/?embryosshowedabnormalitiesthatcouldnotbedetectedintheuntreatedmice.49Theselateembryonicdefectsincludedthymichypoplasia,athin-walledventricularseptum,apoorlydevelopedmandible,otherdevelopmentaldefectsinstructuresderivedfromthecephalicneuralcrestcells,renalhypoplasia,afailuretoformthemetanephros,andanaberrantelongationofthenephricductalongtheantero-posterioraxisoftheembryo.49–51Thedefectofthenephricduct,whichconsistedofanabnormalmorphogenesisandguidanceinthede-velopingkidney,wascharacterizedbythelossofRetexpressionthatisanessentialcomponentoftheglialcell–derivedneurotrophicfactor(GDNF)signalingpathwayinvolvedinuretericbudforma-tionandnephricductguidance.50Thus,Gata3hasaroleinthedifferentiationofmultiplecelllineagesduringembryogenesisaswellasbeingakeyregu-latorofnephricductmorphogenesisandguidanceofthenephricductinitscaudalextensioninthepro/mesonephrickidney.49,50
ItisimportanttonotethatHDRpatientswithGATA3haploinsuf?ciencydonothaveimmunede-?ciency,andthissuggeststhattheimmuneabnor-malitiesobservedinsomepatientswith10pdele-tionsaremostlikelytobecausedbyothergeneson10p.Similarly,thefacialdysmorphism,growth,anddevelopmentaldelay,commonlyseeninpatientswithlarger10pdeletions,wereabsentintheHDRpatientswithGATA3mutations,furtherindicatingthatthesefeatureswerelikelyduetoothergeneson10p.39ThesestudiesofHDRpatientsclearlyindi-cateanimportantroleforGATA3inbothparathy-roiddevelopmentandtheetiologyofhypoparathy-roidism.
AutosomalformsofhypoparathyroidismandGCMBGCMB,whichisthehumanhomologueoftheDrosphiliagenegcm,andofthemouseGcm2gene,isexpressedexclusivelyintheparathyroidglands,indicatingthatitmaybeaspeci?cregulatorofparathyroidglanddevelopment.12–15,52Gcm2?/?micelackedparathyroidglandsanddevelopedthehypocalcemiaandhyperphosphatemiaasobservedinhypoparathyroidism.52,53However,despitetheirlackofparathyroidglands,Gcm2?/?micedidnot
haveundetectableserumPTHlevels.52,53Thisen-dogenouslevelofPTHintheGcm2?/?micewastoolowtocorrectthehypocalcemia,butexogenouscontinuousPTHinfusioncouldcorrectthehypocal-cemia.52Interestingly,therewerenocompensatoryincreasesinPTHrPor1,25(OH)2vitaminD3.These?ndingsindicatethatGcm2?/?micehaveanormalresponse(andnotresistance)toPTH.Long-termtreatmentoftheGcm2?/?micewith1,25(OH)2vitaminD3restoredtheserumcalciumconcentra-tionstonormalandreducedtheserumPTHlevels,therebyindicatingthattheproductionofPTHcanbedownregulated.52ThisGcm2-independentauxil-iarysourceofPTHproductionhasbeenshowntobefromthemedullarythymicepithelialcells(mTECs),inwhichPTHisexpressedasaself-antigenforneg-ativeselection.53
Thespeci?croleofGcm2inthedevelopmentoftheparathyroidsfromthethirdpharyngealpouchhasbeenfurtherinvestigatedbystudyingtheexpressionoftheHoxa3-Pax1/9-Eya1transcrip-tionfactorandShh–bonemorphogeneticprotein4(Shh-Bmp4)signalingnetworks.11Thesestud-ieshaverevealedthatGcm2expressionbeginsatE9.5inthedorsalanteriorpharyngealendo-dermofthethirdpouchandismaintainedinthepresumptivemouseparathyroiddomainatlaterstages,54andthatatE12.0Gcm2?/?embryoshaveaparathyroid-speci?cdomain,butthatthisparathy-roiddomainundergoescoordinatedprogrammedcelldeath(apoptosis)byE12.5intheGcm2?/?mouseembryos.11Moreover,theexpressionofthetranscriptionfactorsHoxa3,Pax1,Pax9,Eya1,andTbx1,andofShhandBmp4,wasnormalinthethirdpharyngealpouchesoftheseGcm2?/?mouseembryos.These?ndingsindicatethattheHoxa3-Pax1/9-Eyatranscriptionfactorcascade,aswellasthetranscriptionfactorTbx1andtheShh-Bmp4signalingnetwork,allactupstreamofGcm2.11In-deed,ithasbeenshownthatHoxa3isrequiredfortheinitiationofGcm2expressioninthethirdpouchendoderm,andbothHoxa3andPax1arere-quiredforthemaintenanceofGcm2expression.55Moreover,thesestudieshaverevealedthatGcm2hasaroleinpromotingdifferentiationandsurvivalofparathyroidcellsinthedevelopingembryo.11Thus,Gcm2isrequiredforthedifferentiationofparathyroidprecursorcellsintheparathyroidspe-ci?cdomain,butisnotrequiredforinitialpat-terningorexpressionofdifferentiationmarkers,
c2011NewYorkAcademyofSciences.Ann.N.Y.Acad.Sci.1237(2011)24–38??31
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suchas,thecalciumsensingreceptor(CaSR)inthecommonparathyroid/thymusprimordia.11Thetar-getgenesofmammalianGCMB/Gcm2arelargelyunknown.However,recentstudiesthatutilizedcul-turedprimaryparathyroidcellsfromhyperplasticglandsofpatientswithchronickidneydisease56havedemonstratedthatdownregulationofGCMBexpression,achievedbyinfectionwithlentivirusex-pressingshRNAforGCMB,resultedindownregu-lationofCaSRexpression,therebysuggestingoneofthefunctionsofGCMBtobemaintenanceofhighlevelsofCaSRexpressioninparathyroidcells.56These?ndingsaresupportedbystudiesincotrans-fectedHEK-293,inwhichexogenousGCMBwasabletotransactivatereporterconstructsthatcon-tainedCaSRpromoterDNAsequencesthatencom-passedGCMBresponseelements.57
Studiesofpatientswithisolatedhypoparathy-roidismhaveshownthatGCMBmutationsareas-sociatedwithautosomalrecessiveanddominantformsofthedisease.58–60,61,62Thus,ahomozygousintragenicdeletionofGCMBhasbeenidenti?edinapatientwithautosomalrecessivehypoparathy-roidism,58whereas,inotherfamiliesahomozy-gousmutation(Arg47Leu)oftheDNA-bindingdomainhasbeenreported.59Recently,fourdiffer-enthomozygousgermlinemutationswereidenti-?edineightfamiliesthatoriginatefromtheIndiansubcontinent(Arg39Stop,Arg47Leu,Arg110Trp,andaframeshiftingdeletion[I298fsX307]).61Func-tionalanalysisusingsubcellularlocalizationstud-ies,electrophoreticmobilityshiftassays(EMSAs),andluciferase-reporterassaysdemonstratedthattheArg39Stopmutantfailedtolocalizetothenucleus;theArg47LeuandArg110TrpmutantsbothlostDNA-bindingabilityandtheI298fsStop307mutanthadreducedtransactivationalability.61Morere-cently,threeheterozygousGCMBmutations,whichconsistofsinglenucleotidedeletions(c1389deTandc1399delC)thatintroduceframeshiftsandprematuretruncations,havebeenidenti?edintwounrelatedfamilieswithautosomaldominanthypoparathyroidism,60andamissensemutationAsn502Hiswasidenti?edinonefamilywithauto-somaldominanthypoparathyroidism.62Thesethreemutationswereshown,byusingaGCMB-associatedluciferasereporterassay,toinhibittheactionofthewild-typetranscriptionfactor,therebyindicatingthattheseGCMBmutantshavedominant-negativeproperties.60,62
X-linkedrecessivehypoparathyroidismandSOX3X-linkedrecessivehypoparathyroidismhasbeenre-portedintwomultigenerational,relatedkindredsfromMissouri,U.S.12–15,63,64Thisdisorderonlyaf-fectsmales,inwhichtheysufferfrominfantileon-setofepilepsyandhypocalcemiathatisduetoanisolateddefectinparathyroidglanddevelopment.65StudiesutilizingX-linkedpolymorphicmarkersinthesefamilieslocalizedthemutantgenetochro-mosomeXq26–q27,66andamoleculardeletional–insertionthatinvolveschromosome2p25andXq27hasbeenidenti?ed.67Thisdeletion–insertionislo-catedapproximately67kbdownstreamofSOX3,andhence,itislikelytoexertapositioneffectonSOX3expression.Moreover,SOX3wasshowntobeexpressedinthedevelopingparathyroidsofmouseembryos,andthisindicatesalikelyroleforSOX3intheembryonicdevelopmentoftheparathyroidglands.67SOX3belongstoafamilyofgenesencod-inghigh-mobilitygroup(HMG)boxtranscriptionfactorsandisrelatedtoSRY,thesex-determininggeneontheYchromosome.Themousehomologueisexpressedintheprestreakembryoandsubse-quentlyinthedevelopingCNSthatincludesthere-gionoftheventraldiencephalons,whichinducesde-velopmentoftheanteriorpituitaryandgivesrisetothehypothalamus,olfactoryplacodes,andparathy-roids.67–70Thelocationofthedeletion–insertion～67kbdownstreamofSOX3inX-linkedrecessivehypoparathyroidpatientsislikelytoresultinalteredSOX3expression,asSOX3expressionhasbeenre-portedtobesensitivetoposition—effectscausedbyX-chromosomeabnormalities.71These?ndingspointtoapotentialrolefortheSOX3geneintheem-bryologicaldevelopmentoftheparathyroidglandsfromthepharyngealpouches.
PluriglandularautoimmunehypoparathyroidismandAIRE1HypoparathyroidismmayoccurinassociationwithcandidiasisandautoimmuneAddison’sdisease,andthedisorderhasbeenreferredtoaseitherautoim-munepolyendocrinopathy-candidiasis-ectodermaldystrophy(APECED)syndromeorautoimmunepolyglandularsyndrometype1(APS1).12–15,72ThisdisorderhasahighincidenceinFinland,andage-neticanalysisofFinnishfamiliesindicatedautoso-malrecessiveinheritanceofthedisorder.73Inaddi-tion,thedisorderhasbeenreportedtohaveahigh
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incidenceamongIranianJews,althoughtheoccur-renceofcandidiasiswaslesscommoninthispopu-lation.74LinkagestudiesofFinnishfamiliesmappedtheAPECEDgenetochromosome21q22.3.75Fur-therpositionalcloningapproachesledtotheiso-lationofanovelgenefromchromosome21q22.3.Thisgene,referredtoasAIRE,encodesa545aminoacidproteinthatcontainsmotifssuggestiveofatranscriptionalfactorandincludestwozinc-?ngermotifs,aproline-richregion,andthreeLXXLLmo-tifs.76FourAIRE1mutationsarecommonlyfoundinAPECEDfamilies,andtheseareArg257StopinFinnish,German,Swiss,British,andNorthernItalianfamilies;Arg139StopinSardinianfamilies;Tyr85CysinIranianJewishfamilies;anda13bpdeletioninExon8inBritish,Dutch,German,andFinnishfamilies.76–80AIRE1hasbeenshowntoreg-ulatetheeliminationoforgan-speci?cTcellsinthethymus,andthus,APECEDislikelytobecausedbyafailureofthisspecializedmechanismfordelet-ingforbiddenTcellsandestablishingimmunolog-icaltolerance.81PatientswithAPS1mayalsode-velopotherautoimmunedisordersinassociationwithorgan-speci?cautoantibodies,whicharesim-ilartothoseinpatientswithnon-APS1formsofthedisease.ExamplesofsuchautoantibodiesandrelateddiseasesareGAD6Sautoantibodiesindia-betesmellitustype1Aand21-dydroxylaseautoan-tibodiesinAddison’sdisease.PatientswithAPS1mayalsodevelopautoantibodiesthatreactwithspeci?cautoantigensthatarenotfoundinnon-APS1patients,andexamplesofthisareautoanti-bodiestotype1interferon?,whicharepresentinallAPS1patients,82andtoNACHTleucine-rich-repeat-protein5(NALP5),whichisaparathyroid-speci?cautoantibodypresentin49%ofpatientswithAPS1-associatedhypoparathyroidism.83NALPproteinsareessentialcomponentsofthein?am-masoneandactivatetheinnateimmunesystemindifferentin?ammatoryandautoimmunedisorders,suchas,vitiligo,whichinvolvesNALP1,andgout,whichinvolvesNALP3.84ThepreciseroleofNALP5inAPS1-associatedhypoparathyroidismremainstobeelucidated.Aire1?/–micemimickingthecom-monhuman13-basepairdeletionmutationpresentwithonlyamildautoimmunephenotype,withanevidentincreaseinthenumberofactivatedTcellsanddetectionofautoantibodiesagainstseveralor-gans.85Atthehistologicallevel,lymphocyticin-?ltrationofseveralorgansindicatedthedevelop-
mentofautoimmunity,althoughsymptomsweremildandthequalityoflifeforAire1?/?miceap-pearedequivalenttowild-typelittermates,withtheexceptionofmaleinfertility,suggestingthataddi-tionalgeneticand/orenvironmentalfactorscon-tributesubstantiallytotheovertnatureofautoim-munityassociatedwithAire1mutations,evenformutationsidenticaltothosefoundinhumanswithAPECED.85
Transcriptionfactordefectsassociatedwithparathyroiddevelopmentalabnormalitiesinmice
Deletionsofgenesencodingforseveraltran-scriptionfactors,inmice,resultindevelopmen-talanomaliesthatincludeparathyroiddefects,al-thoughsuchabnormalitieshavenotasyetbeenreportedinman(Table1).12–15Thesetranscrip-tionfactorsincludemembersofthehomeobox(Hox)andpairedbox(Pax)families,theeyesabsenthomologue(Eya1)andsineoculishome-oboxhomologue(Six1;Table2).Hoxgenesspec-ifypositionalidentityinthedevelopingembryoandplayrolesintheformationandpatterningoftheposteriorpharyngealpouchesandtheirorganderivatives.86Forexample,Hoxa3isstronglyex-pressedinthethirdpharyngealarch,third,andfourthpharyngealpouchendoderm,andneu-ralcrestmesenchyme,andHoxa3?/?micede-velopseveredefectsinpharyngealorgandevelop-mentthatincludesaplasiaoftheparathyroidsandthymus,thyroidhypoplasia,andneural,skeletal,andcardiovasculardefects,8,55,87,88whereascom-poundHoxa3+/?Hoxb3?/?,andHoxd3?/?micedevelopectopicthymusandparathyroids.8Simi-larly,Pbx1?/?mice,whichlackPbx1,athreeaminoacidloopextension(TALE)classhomeodomainproteinthatactsasacofactorofHOXtranscrip-tionfactors,developpharyngealregionabnormal-itiesthatincludeabsentorhypoplasticparathy-roid/thymusprimordia(Table2).89
ThePax1/9-Eya1-Six1networkhasbeeniden-ti?edtoactdownstreamofHoxa3duringpat-terningandearlyorganogenesisofboththethy-musandparathyroids.5,55,90–95Pax1,whichislikelytobedownstreamofHoxa3,isexpressedinthepharyngealendoderm,96andPax1?/?micede-velopparathyroidandthymicdysplasia.55,92ThesePax1?/?micehaveupregulationofPax9,whichhasahighsimilaritytothepeptidesequenceofPax1,91
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