Ann.N.Y.Acad.Sci.ISSN0077-8923
ANNALSOFTHENEWYORKACADEMYOFSCIENCES
Issue:SkeletalBiologyandMedicineI
Transcriptionfactorsinparathyroiddevelopment:lessonsfromhypoparathyroiddisorders
IrinaV.GrigorievaandRajeshV.Thakker
AcademicEndocrineUnit,Nuf?eldDepartmentofClinicalMedicine,OxfordCentreforDiabetes,Endocrinology,andMetabolism(OCDEM),ChurchillHospital,UniversityofOxford,Headington,Oxford,UnitedKingdom
Addressforcorrespondence:RajeshV.Thakker,AcademicEndocrineUnit,Nuf?eldDepartmentofMedicine,OxfordCentreforDiabetes,Endocrinology,andMetabolism(OCDEM),ChurchillHospital,UniversityofOxford,Headington,Oxford,OX37LJ,UK.rajesh.thakker@ndm.ox.ac.uk
Parathyroiddevelopmentalanomalies,whichresultinhypoparathyroidism,arecommonandmayoccurinonein4,000livebirths.Parathyroids,inman,developfromtheendodermalcellsofthethirdandfourthpharyn-gealpouches,whereas,inthemousetheydevelopsolelyfromtheendodermofthethirdpharyngealpouches.Inaddition,neuralcrestcellsthatarisefromtheembryonicmid-andhindbrainalsocontributetoparathyroidglanddevelopment.Themolecularsignalingpathwaysthatareinvolvedindeterminingthedifferentiationofthepharyngealpouchendodermintoparathyroidcellsarebeingelucidatedbystudiesofpatientswithhypoparathy-roidismandappropriatemousemodels.Thesestudieshaverevealedimportantrolesforanumberoftranscriptionfactors,whichincludeTbx1,Gata3,Gcm2,Sox3,Aire1andmembersofthehomeobox(Hox)andpairedbox(Pax)families.
Keywords:hypocalcemia;embryonicdevelopment;congenital;parathyroidhormone
Introduction
Parathyroiddevelopmentalanomaliesresultinhy-poparathyroidism,andstudiesofdisordersasso-ciatedwithhypoparathyroidism(Table1)haverevealedimportantrolesforsometranscriptionfac-torsinembryonicparathyroiddevelopment.Fur-thermore,deletionsofthegenesencodingthesetranscriptionfactorstogeneratemousemodelshavehelpedtorevealthetranscriptionalcascadesthatareinvolved.Inparallel,otherstudiesthathavegeneratedmicedeletedforadditionaltranscriptionfactorsinvolvedinembryonicdevelopment,haveobservedtheoccurrenceofparathyroidaplasiaorhypoplasia,althoughmutationsforthesegenesinpatientswithhypoparathyroidismhaveyettobeidenti?ed.Thisreviewwilldescribethedevelop-mentalandevolutionaryoriginsoftheparathy-roids,thehypoparathyroiddisorders(Table1),andthehumanandmousestudiesthathaverevealedtherolesoftranscriptionfactorsinparathyroiddevelopment.
Parathyroidglandevolutionanddevelopment
Amphibianshaveparathyroidglands,whereas,?shlacktheseglandsandinsteadhavegills,whichde-velopasinvaginationsfromtheectodermalepithe-liumofthethird,fourth,?fth,andsixthpharyn-gealarchesandareconsideredtobeevolutionaryhomologousstructures.1Inmammaliandevelop-ment,theparathyroidglands,togetherwiththethymus,thyroid,andultimobranchialbodies,arederivedfromthepharyngealregion(Fig.1)withcontributionsfromthethirdandfourthpharyn-gealpouchesandcranialneuralcrestcellsthatarisefromtheembryonicmid-andhindbrain.2Inman,therearetwopairsofparathyroidglands,referredtobytheirlocationsinrelationshiptothethyroidassuperiorandinferiorparathyroids.Thesupe-riorparathyroidglandsarederivedfromtheen-dodermofthefourthpharyngealpouches(Fig.1).Theinferiorparathyroidglandsdevelopfromtwocommonparathyroid/thymusprimordia,whichare
doi:10.1111/j.1749-6632.2011.06221.x
24c2011NewYorkAcademyofSciences.Ann.N.Y.Acad.Sci.1237(2011)24–38??
Grigorieva&ThakkerTranscriptionfactorsinparathyroiddevelopment
Table1.Geneticbasisofsomehypoparathyroiddisorders
inmanandparathyroiddevelopmentalabnormalitiesinmice
Mouseknockout
models
Humandisorders
ComplexcongenitaldisorderswithhypoparathyroidismDiGeorgesyndrometypeI(TBX1)HDR(GATA3)APECED(AIRE1)Kenny–Caffey
syndrome(TBCE)KSS,MELAS,MTPDS(mitochondria)Blomstrandlethalchondrodysplasia(PTH/PTHrP)PHPIa,Ib,PPHb(GNAS1)?
Tbx1Gata3Aire1Tbce?Lethal(?)
Gnas1
Hoxa3,Pax1,Pax3,Pax9,Pbx1,Eya1,Six3PthGcm2Sox3Casr
IsolatedhypoparathyroidismPTHGCMB
XLHPT(Xq26-q27;SOX3)CaSR
TBCE,tubulin-speci?cchaperone;KSS,KearnsSayreSyn-drome;MELAS,mitochondrialencephalopathy,stroke-likeepisodes,andlacticacidosis;MTPDS,mitochon-drialtrifunctionalproteinde?ciencysyndrome;PTHrP,parathyroidhormone-relatedprotein;GNAS1,geneen-codingforthe?subunitoftheGprotein,Gs,whichcou-plesreceptorbindingbyseveralhormonestoactivationofadenylatecyclase;?,unknown.
derivedfromendodermalcellsofthethirdpha-ryngealpouches.Laterindevelopment,theinfe-riorglandsseparatefromthethymusandcometoliecaudaltothethyroidandsuperiorparathyroidglands.Inmice,thereisonlyonepairofparathy-roidglands,whichoriginatefromtheendoderm-derivedepitheliallayerthatlinesthethirdpharyn-gealpouches;thisishomologoustotheinferiorparathyroidglandsinman.Inadditiontotheen-dodermalcellsofthethirdandfourthpharyngealpouches,cellsoriginatingfromtheneuralcrestof
rhombomeres6and7ofthehindbrainalsocon-tributetotheanlageoftheparathyroidglands.3Theneuralcrestcellsoriginateattheappositionofneu-roectodermandectodermduringtheformationoftheneuraltube,4andtheneuralcrestcellsinitiallymigratetowardstheforegutendoderm,afterwhichtheyaddtotheanlageoftheparathyroidglands.Neuralcrestcellsofrhombomere6migratetowardthethirdpharyngealarch,whereasthefourthpha-ryngealarchisprimarilyinvadedbyneuralcrestcellsfromrhombomere7.5Inmice,thethirdpha-ryngealpouchesbecomevisiblebyembryonicday(E)9.5–10,andfromE10.5thesedifferentiatetoformtheirderivatives.6,7Anepithelialoutgrowthofeachthird-pouchendodermbeginstoformthebilateralparathyroid/thymuscommonprimordia,whicharedividedintospeci?cdorsalandanteriorpresumptiveparathyroiddomainsandventralanddistalpresumptivethymusdomains.Theparathy-roidandthymuscanberecognizedatE12.5–13,8andseparationoftheparathyroidglandsfromthethymusoccursbyE14–14.5,whenbothstructuresmigratetowardsthecaudalend.9,10Theparathyroidglandsbecomesituatedposteriortothelobesofthethyroid,whereas,thethymusdescendsfurtherinthedirectionoftheheart.Thus,theearlystagesofparathyroidorganogenesisarecloselylinkedwiththymusorganogenesis.Atthisstage,theparathy-roidglandsshowahighdegreeofvascularity,andtheirinnervation,aswellasthethyroidandthethy-mus,isderivedfromthecervicalsympatheticgan-gliaandbranchesofthevagusnerve.2Expressionoftheparathyroidhormone(Pth)geneoccursasearlyasatE11.5,beforeseparationfromthethy-mus,andismaintainedthroughoutdevelopment.11Thus,theformationoftheparathyroidglandsinmicecanbeseparatedintofourdifferentstagesthatinclude(1)earlypatterningofthethirdpharyngealpouch,(2)speci?cationoftheparathyroiddomainintheparathyroid/thymusprimordia,(3)migra-tionoftheparathyroidglandstowardstheir?naldestination,and(4)furtherdifferentiationtowardsPTH-producingcells.Themolecularmechanismsthatareinvolvedinthesestageshavebeeneluci-datedfromstudiesofmicedeletedforspeci?cgenesandfromstudiesinpatientswithhypoparathy-roidism,andsomeofthesegenesencodetranscrip-tionfactorsthatincludeTbx1,Gata3,Gcm2,Sox3,Aire1,Hoxa3,Pbx1,Pax1,Pax3,Pax9,Eya1,andSix1(Table2).
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TranscriptionfactorsinparathyroiddevelopmentGrigorieva&Thakker
Figure1.(A)Schematicdiagramofthetransverseviewofthepharyngealregioninamammalianembryoshowingtheendodermalpharyngealpouchesandmesodermalpharyngealarches.Thepharyngealendodermofthethirdandfourthpouchesgivesrisetotheparathyroidglandsandthymusinthehumanembryoduringthe?fthweekofgestation,whereas,inthemouse,theparathyroidsarederivedfromthethirdpouchduringembryonic(E)days10.5–12.5.(B)SchematicdiagramofthesagittalviewofthepharyngealregionofanE10.5mouseembryoshowingthemigrationoftheneuralcrestcells(dottedarrows)fromtherhombomeres(r)ofthehindbraintothepharyngealarches.
Hypoparathyroiddisordersduetotranscriptionfactordefects
Hypoparathyroidismmayoccuraspartofapluriglandularautoimmunedisorderorasacom-plexcongenitaldefect,asforexample,inDiGe-orgesyndrome(DGS;Table1).12–15Inaddition,hypoparathyroidismmaydevelopasasolitaryen-docrinopathy,andthishasbeencalledisolatedoridiopathichypoparathyroidism.Familialoccur-rencesofisolatedhypoparathyroidismwithautoso-maldominant,autosomalrecessive,andX-linkedrecessiveinheritanceshavebeenestablished.Stud-iesoftheseformsforhypoparathyroidismhavere-vealedthatdefectsintranscriptionfactorsarein-volvedintheetiologyofthesedisordersandinparathyroiddevelopment(Tables1and2).12–15
DiGeorgesyndromeandTBX1PatientswithDGStypicallysufferfromhy-poparathyroidism,immunode?ciency,congenitalheartdefects,anddeformitiesoftheear,nose,andmouth.12–16Thedisorderarisesfromacongenitalfailureinthedevelopmentofthederivativesofthethirdandfourthpharyngealpoucheswithresult-ingabsenceorhypoplasiaoftheparathyroidsandthymus.MostcasesofDGSaresporadic,butanautosomaldominantinheritanceofDGShasbeenobservedandanassociationbetweenthesyndromeandanunbalancedtranslocationanddeletionsin-
volving22q11.2havealsobeenreported17—thisisreferredtoasDGStype1(DGS1).Insomepatients,deletionsofanotherlocusonchromosome10phavebeenobservedinassociationwithDGS,andthisisreferredtoasDGStype2(DGS2).18MappingstudiesoftheDGS1deletedregiononchromosome22q11.2de?neda250–3000kbcriticalregion19,20thatcontainedapproximately30genes.StudiesofDGS1patientshavereporteddeletionsofseveralofthegenes(e.g.,rnex40,nex2.2–nex3,UDFIL,andTBX1)fromthecriticalregion,17,21–23andstudiesoftransgenicmicedeletedforsuchgenes(e.g.,Udf1l,Hira,andTbx1)haverevealeddevelopmentalab-normalitiesofthepharyngealarches.24–26However,pointmutationsinDGS1patientshaveonlybeendetectedintheTBX1gene(TBX1),27andTBX1isnowconsideredtobethegenecausingDGS1.28TBX1isaDNA-bindingtranscriptionalfactor,oftheT-boxfamily,thatisknowntohaveanimpor-tantroleinvertebrateandinvertebrateorganogene-sisandpatternformation.TBX1isdeletedin～96%ofDGS1patients.Moreover,DNAsequenceanal-ysisofunrelatedDGS1patients,whodidnothavedeletionsofchromosome22q11.2,revealedtheoc-currenceofthreeheterozygouspointmutations.27Oneofthesemutationsresultedinaframeshiftwithaprematuretruncation,whereas,theothertwoweremissensemutations(Phe148TyrandGly310Ser).Allofthesepatientshadthecompletepharyngealphenotypebutdidnothavementalretardationor
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Grigorieva&ThakkerTranscriptionfactorsinparathyroiddevelopment
learningdif?culties.Interestingly,transgenicmicewithdeletionofTbx1haveaphenotypethatissim-ilartothatofDGS1patients.26Thus,Tbx1nullmutantmice(Tbx1?/?)hadallthedevelopmentalanomaliesofDGS1(i.e.,thymicandparathyroidhy-poplasia,abnormalfacialstructuresandcleftpalate,skeletaldefects,andcardiacout?owtractabnormal-ities),whereas,Tbx1haploinsuf?ciencyinmutantmice(Tbx1+/?)wasassociatedonlywithdefectsofthefourthpharyngealpouch(i.e.,cardiacout?owtractabnormalities).Tbx1inmiceisexpressedinthepharyngealectodermandendoderm,aswellasinthemesodermalcoreofthepharyngealarches,butisnotexpressedinneuralcrestcells.29DetailedstudiesinthesemicehaverevealedthatinactivationofTbx1resultsinabnormalearlypatterningandhy-poplasia/aplasiaofthepharyngealarches,aswellasimpairedformationofthesecondandfourthpharyngealpouches.26,30,31Furthermore,inducibleinactivationofTbx1inconjunctionwithinvivocell-fatemappinghasdemonstratedthattheearlyabsenceofTbx1inE7.5embryosresultedinaphenotypeidenticaltothatobservedingermlineTbx1?/?knockoutmice.Thus,inactivationofTbx1ataroundE9.0exclusivelyimpairedthedevelop-mentofpharyngealsegmentsposteriortoandin-cludingthethirdpharyngealpouch.Thisindicatesthatthereisalikelyanterior-to-posteriorgradientofTbx1activityovertimeandthatTbx1expres-sionistightlyregulatedindistinctsegmentsduringpharyngealsystemdevelopment.32cDNAmicroar-rayanalysesofmicelackingTbx1haveidenti?edthetranscriptionfactorglialcellsmissing2(Gcm2)asoneofthedownregulatedgenesinthepharyngealregion,33therebyindicatingthatTbx1isupstreamofGcm2.Moreover,Tbx1isregulatedbysonichedge-hog(Shh),therebyindicatingaShh-Tbx1-Gcm2pathwayinparathyroiddevelopment.11,34
ThebasisofthephenotypicdifferencesbetweenDGS1patients—whoareheterozygous—andtheTbx1+/?miceremainstobeelucidated.Itisplau-siblethatTbx1dosage,togetherwiththedown-streamgenesthatareregulatedbyTbx1,couldpro-videanexplanation,buttherolesoftheseputativegenesinDGS1remainstobeelucidated.Somepatientsmayhavelate-onsetDGS1,andthesein-dividualsdevelopsymptomatichypocalcemiainchildhoodorduringadolescencewithonlysub-tlephenotypicabnormalities.35,36Theselate-onsetDGS1patientshavesimilarmicrodeletionsinthe
22q11region.ItisofinteresttonotethattheageofdiagnosisinthefamiliesofthethreeDGS1patientswithinactivatingTBX1mutationsrangedfrom7to46years,whichisinkeepingwithlate-onsetDGS1.27
Hypoparathyroidism,deafness,andrenalanomaliessyndromeandGATA3Thecombinedinheritanceofhypoparathyroidism,deafness,andrenaldysplasia(HDR)asanautoso-maldominanttraitwasreportedinonefamilyin1992.12–15,37,38Patientshadasymptomatichypocal-cemiawithundetectableorinappropriatelynormalserumconcentrationsofPTHandnormalbriskin-creasesinplasmacAMPinresponsetotheinfusionofPTH.Thepatientsalsohadbilateral,symmet-rical,andsensorineuraldeafnessinvolvingallfre-quencies.Therenalabnormalitiesconsistedmainlyofbilateralcyststhatcompressedtheglomeruliandtubulesandleadtorenalimpairmentinsomepa-tients.Cytogeneticabnormalitieswerenotdetected,andabnormalitiesofthePTHgenewereexcluded.37However,cytogeneticabnormalitiesinvolvingchro-mosome10p14–10pterwereidenti?edintwoun-relatedpatientswithfeaturesthatwereconsistentwithHDR.Thesetwopatientssufferedfromhy-poparathyroidism,deafness,andgrowthandmentalretardation;onepatientalsohadasolitarydysplas-tickidneywithvesico-uretericre?uxandauterusbicornisunicollis,andtheotherpatient,whohadacomplexreciprocal,insertionaltranslocationofchromosomes10pand8q,hadcartilaginousexos-toses.39Neitherofthesepatientshadimmunode-?ciencyorheartdefects,whicharekeyfeaturesofDGS2(seeearlier),andfurtherstudiesde?nedtwononoverlappingregions;thus,theDGS2regionwaslocatedon10p13–14andHDRon10p14–10pter.DeletionmappingstudiesintwootherHDRpa-tientsfurtherde?nedacritical200kbregionthatcontainedGATA3,39whichbelongstoafamilyofzinc-?ngertranscriptionfactorsthatareinvolvedinvertebrateembryonicdevelopment.DNAsequenceanalysisinotherHDRpatientsidenti?edmuta-tionsthatresultedinahaploinsuf?ciencyandlossofGATA3function.39–43GATA3,whichisa444aminoacidprotein,hastwozinc?ngers—theC-terminal?nger(ZnF2)bindsDNA,whereastheN-terminal?nger(ZnF1)stabilizesthisDNAbindingandinteractswithotherzinc-?ngerproteins,suchas,theFriendsofGATA(FOG).44HDR-associated
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TranscriptionfactorsinparathyroiddevelopmentGrigorieva&Thakker
Table2.Sometranscriptionfactorsinvolvedinmouseparathyroidglanddevelopment
Transcriptionfactor(TF)Hoxa3
Siteofexpression
Positionintheparathyroidgeneticpathway
Phenotypeofnull(?/?)mouse
Mutations(human)
FunctionHomeodomainTF
Reference5,8,87,88,112
Pbx1
EarlyexpressioninDownstreamof:
retinoicacidthe3rdand4th
Upstreamof:pharyngealarches,
Pax1/9,Eya1,pharyngeal
Six1/4,Gcm2endoderm,and
neuralcrestmesenchyme
[unknown]ExpressionatTALEclass
E9.5–10.5ishomeodomainTF.
presentinsurfaceCofactorofHox
ectoderm,TFs
pharyngealpouchendoderm,subsetsofarch
mesenchyme
NonereportedFailureofinitiationof
parathyroid/thymusprimordialformationaswellasthyroidhypoplasia
Pax1Paired-boxTF
Pax3
Pax9
Eya1
DorsalshiftintheborderWaardenburg
syndrome(typesI;betweenparathyroid-III)characterizedandthymusdomains
bydeafnessandatE11.5,resultingin
pigmentationlargerthymic
anomaliesrudimentsandsmaller
parathyroidrudimentsbyE12.5
Absenceofparathyroid,ToothagenesisPaired-boxTFEarlyexpressioninDownstreamof:
(molarthymus,andTbx1pharyngeal
oligodontia)Upstreamof:Eya1,ultimobranchialendodermand
bodies.TheepithelialBmp4mesoderm
budsseparatingfromthe3rdpharyngealpouchdonotforminmutantmice
FromE9.5,theorganDownstreamof:ExpressionstartsTFwithconservedAutosomaldominantprimordiaforbothHoxa3,Pax1,Pax9fromE9.5inEyadomain(ED),Branchio-oto-parathyroidandUpstreamof:pharyngealarchespartofproteinrenal(BOR)thymusfailedtoformSix1,Gcm2,Tbx1,(1–4),pouchtyrosinesyndrome
–/–inEya1Fgf8endoderm(2–4)phosphatefamilycharacterizedbyembryos.
inallthreecell(PTP)hearingloss,Thyroidhypoplasia,types:neuralbranchialcysts,withseverereductioncrest–derivedarchandrenalfailureinthenumberofmesenchyme,parafollicularcellsandpouchendoderm,sizeofthyroidlobes,andsurfacelackoffusionbetweenectodermfromtheultimobranchialE9.5bodiesandthethyroid
lobePaired-boxTF
[unknown]
Expressionstartsintheearlyendodermal
epitheliumofthe3rdpharyngealpouch
Neuralcrestcells
NonereportedDelayedorabsent
formationofcaudalpharyngealpouches;disorganized
patterningofthe3rdpharyngealpouchresultinginabsentorhypoplastic
parathyroid/thymusprimordial
HypoplasticparathyroidNonereportedDownstreamof:
andthymus,disturbedHoxa3
Upstreamof:Eya1,thymocytematuration.Gcm2
89
55,92
101
91,93,102
94,95,113
Continued
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