CalcaneusOssificationanditsRelationshiptoGeneExpression

inComparisontoPisiformFormation

Abstract

Theexpansionofevolutionaryresearchreliesonstudiesthatfurtheranunderstandingof

theinfluenceofgeneticfactorsonphenotypethroughdevelopmentalprocesses.Inmany

mammalianspecies,thepisiformandcalcaneusaretwouniquebonesthatformgrowthplates.

However,thereasonforgrowthplateformationinthesebonesaswellasthefactorsinvolved

withthisprocessareunknown.Inthisproject,wewillobservetheformationofthecalcaneusin

miceandcomparethetimingandpatterningtopisiformformation.Additionally,theHoxgene

expressionpatternsinthecalcaneuswillbeobservedfortheeffectHoxexpressionhason

growthplateformationaswellashowitcomparestocharacteristicsofpisiformformation.This

projectaimstorevealtheunderlyingmechanismsinvolvedwithcalcaneusdevelopmentand

potentialrelationshipsbetweencalcaneusandpisiformgrowthplateformation.

Introduction:BoneDevelopment

Manybonesinthebodyareformedthroughgrowthplateformation.Theformationofthe

primaryandsecondaryossificationcentersduringbonedevelopmentisnecessaryforgrowth

plateformation.Thisprocessbeginswithcondensationofmesenchymalcells,leadingtochanges

inthesecellstobecomechondrocytes.ThesechondrocyteswillsecretetypeIIcollagenand

proliferatetomakeupacartilaginousmatrix.Eventuallychondrocytesinthecenterofthematrix

regulatematrixmineralizationandrecruitmentofosteoblasts.Cellsinthematrixwillthendie

andbereplacedbyamatrixofbonecellslaiddownbyosteoblasts.Thisfirstoccursatthe

primarycenterofossification.Thechondrocytesaroundthiscenterorganizethemselvesand
allowforthelengtheningofthebone.Migrationofchondrocytesmayleadtoformationofa

secondaryossificationcenter,anoccurrencecommonlyseeninlongbones.Thesecondarycenter

isestablishedwhenchondrocytesstopproliferatingandrecruitosteoblastsforossification.The

cartilagebetweentheprimaryandsecondarycentersisknownasthegrowthplate.Thegrowth

plateisorganizedwithregionsofcellsproliferatingrapidly,expanding(hypertrophy),and

ossifying,allowingfortheformationofboneandeventuallybridgingthegapbetweenthetwo

centers(Kronenberg,2003).

Formationofabonethroughgrowthplateformationinvolvesalargenumberof

regulatoryfactors,especiallyinrelationtochondrocyteactivity.Anexampleofthenumerous

signalsinvolvedwithboneformationisthecommunicationbetweenIndianhedgehog(Ihh)

pathwaysandparathyroidhormonerelatedproteins(PTHrPs).Ihhpathwaysregulate

chondrocyteproliferationanddifferentiationindevelopingbone.Ihhalsostimulatesconversion

ofperichondrialcellsintoosteoblasts.PTHrPsfunctiontomaintainchondrocyteproliferation.

InteractionsbetweenPTHrPandIhhsignalinginfluencechondrocyteactivityinthegrowthplate,

determiningwhenchondrocytesproliferateandwhentheyundergohypertrophy.Thetimingand

locationofthechangesinchondrocyteactivityaffectthelengthandcharacteristicsofthebone

(Kronenberg,2003).

Manyfactorsandcellsignalingpathwaysplayaroleinlimbformation.Notonlydothese

factorscreatetheprocessesinvolvedwithboneformation,butthespatialarrangementof

differentiatingtissuesisaffectedaswell.Thisprocessofpatternformationisnecessaryforlimb

development.Signalinginpatternformationallowsforcellstoknowtheirlocationandspatial

relationshiptooneanotheranddifferentiateaccordingtothatinformation.Establishmentof
proximalanddistalfeaturesaswellasdifferentialmedialandlateralformationsoccursthrough

geneexpression,influencedbysignalingandregulatoryfactors(Gilbert,2004).

Theestablishmentofgeneregulatoryfactordomainsandthedifferentialeffectsthese

factorshaveongeneexpressionaretheunderlyingmechanismsoflimbpatternformation.Hox

genedomainsandtheireffectsplayacrucialroleinpatternformationwithinalimb.Whilethere

aresimilarfactorsinvolvedwiththeformationoftheforeandhindlimbsthatconstrainlimb

evolution,differinginfluencesontheseprocessesexist,allowingfordifferentialfunctionofthese

limbs(Gilbert,2004).Theconnectionbetweenthemechanismsoflimbdevelopmentandtheir

effectsstillneedsfurtherexpansion,providingthecoremotivationofourresearch.Expanding

knowledgeoftheprocessesinvolvedinlimbdevelopmentisnecessaryforagreater

understandingofvertebratedevelopmentaswellasthewaybywhichthesefactorsshapeand

havebeenshapedbyevolution.

Introduction:FormationofCalcaneusandPisiform

Whiletheprocessofgrowthplateformationiscommoninlongbones,manyotherbones

donotformgrowthplates.Theseincludemostofthetarsalsandcarpals.Mostchondrocytesin

thewristdonotorganizethemselvesorexhibitexpectedgeneexpressionpatternsindicativeof

growthplateformation(Koyama,2010).However,thepisiformontheforelimbandthe

calcaneusonthehindlimbformsecondarycentersofossificationandexhibitgeneexpression

similartogrowthplatesofnearbylongbones.Thissuggeststhattheyformgrowthplatesaspart

oftheirdevelopment.

Micewillbeusedasamodelforexaminationofthisdevelopmentbecausemanylitters

canbebredeasilyandstudiedatspecificages,bothatparticularembryologicalagesandinthe
firstdaystoweeksoflife.Althoughthestudyofprimateswouldseemidealforcomparisonwith

humandevelopment,morphologicalintegrationpatterninginthelimbshasbeenfoundtobe

similarbetweenmiceandprimates(Hallgrimsson,2002).Theconservationoftheunderlying

mechanismsofthispatterningallowsformicetobeanaptmodelforthestudyofthemammalian

appendicularskeleton.Additionally,thesegrowthplatesareformedinthepisiformand

calcaneusofmice,andthusmiceprovideareliablerepresentationofforeandhindlimb

formationinmostmammals.

Humans,however,exhibitwristmorphologythatdiffersfromothermammalspecies.

Reductionofthepisiformsuggeststhatgrowthplateformation,andthusthefactorsthat

contributetothisprocessinthehumanpisiform,ismodifiedinthehumanlineage(Kjosness,

2014).Withtheshifttobipedalism,agreatchangeinhindlimbdevelopmentaccompaniedthis

noticeablechangeinforelimbdevelopment.Thisisreflectedinobservablechangesinanklebone

development.Althoughthehumancalcaneusdiffersmorphologicallyfromthoseofother

mammals,growthplateformationisretained.

Theanklemorphologyofchimpanzees,especiallythetibiaandtalus,reflectstheir

verticalclimbingability.Examinationofearlyhomininsshowsthattheylackedthegreat

dorsiflexionandfootinversionabilitiesofmodernchimps(DeSilva,2009).Thebipedalfoot

functionsinbothpropulsionandenergydissipation.Comparisonofthecalcaneusofmodern

Africanapesandhumansrevealsdifferencesinmorphologyrelatedtotheirpreferredtypesof

locomotion.A.afarensis,incomparisontothesecloselyrelatedmodernspecies,wasfoundto

havemanyofthecalcanealcharacteristicsnecessaryforfunctionsinprolongedbipedal

movement(Latimer,1989).
 Theremarkable,yetconfounding,factaboutthecalcaneusisthelargeeffectenvironment

(esp.loading)canhaveonitsmorphology.Thesechangescanbothaffectmorphologyinan

individualandshapetheadaptabilityoffutureheritabletraits.Whilechangesindevelopmental

morphologyaredirectlyinfluencedbygeneticprocesses,evidencesupportsthatstrainonthe

calcaneushasresultedinmodificationofthecalcaneus,notonlyshapingthebonethroughout

usebutalsopossiblycreatingaselectiveinfluenceforchangesintheheritablemechanismsof

calcaneuspatternformation(Skedros,2004).

Theshifttowardsextendedbipedalloadingonthecalcaneusaswellasashiftinwrist

morphologyoccurredinthehumanlineage,resultinginmorphologythatdiffersfromthatof

othermammalianspecies.Thewaytheseeventsrelatetooneanotherandthefactorsthat

influencedthemhavenotbeenstudiedindepth,creatinganecessityforthestudyofcalcaneus

developmentandthefactorsthatinfluencecalcanealossification.

Introduction:HoxGenes

Hoxgenesarecisregulatoryfactorsthatareresponsibleforregulatinggeneexpressionin

developmentofanimals.Hoxgenesarefactorsthatdonotdirectlycontrolcellfunctionbuthave

aregulatoryinfluenceongenes,theexpressionofwhichdeterminestheactionsandroleofacell.

Whiletheseancestralgenesarefoundacrossmanyspecies,duplicatesoftheseconserved

complexeshaveevolvedinmammals,creatingmultiplegeneparalogues.WhileDrosophila

containasinglesetofeightHoxgenes,mammalshavefourHoxcomplexesknownasHoxA

throughHoxDthateachcontainupto13genes.Hoxgenesareexpressedinparticularphases

anddomains,establishingdevelopmentalfields.Thesefieldsareanecessarypartoftheprocess

ofpatternformationthroughoutadevelopingindividual(Lovejoy,2003).
 Hoxgenesfromsimilarancestralgenesplayrolesinsimilarpartsoforganism

development(Lovejoy,2003).TimingandexpressionofcertainHoxgenesestablisheslimb

patterning,thusinfluencing,ifindirectly,patternsofcellproliferation,differentiation,and

ossificationduringlimbboneformation.Thestudyofthesegenescouldrevealdifferentialor

similarmechanismsthataffectcalcaneusandpisiformformationaswellasthepresenceorlack

ofgrowthplates.

Hox13playsanimportantroleinautopodformation.CompleteknockoutofHox13

homologsinmiceresultsinlossoftheautopod(Gilbert,2013).InanexperimentbyFromental

Ramain,etal.,disruptiveHoxa13heterozygousandHoxd13homozygousmutationsaffectedthe

phalanges,metacarpals,anddistalcarpalsinthemouseforelimb,butdidnotaffecttheproximal

carpals,includingthepisiform.Asimilareffectwasobservedinthehindlimbwithnormal

formationofthetalusandcalcaneus.AlthoughdoublehomozygousmutationsinHoxa13and

Hoxd13resultedinsomealteredcartilageformation,theystillsawcondensationofthepisiform

andcalcaneus(FromentalRamain,1996).Itislikelythattheabilityofthecalcaneusand

pisiformtoformgrowthplatesandproximallocationisconnectedtorelianceon

zeugopodrelatedfactors.

Hox11isknowntoplayaroleinformationoflowerarmandlowerlegbones

(zeugopod).KnockoutsofallHox11paralogspreventszeugopodformationcompletely(Gilbert,

2013).InastudybyDavis,etal.deletionsinHoxa11andHoxd11affectedgrowthofforelimb

longbones(1995).Thismutationhadaminorimpactonhindlimbdevelopmentaswell.This

indicatesoverlapinthefactorsthataffectforeandhindlimbdevelopment.Mutantswith
deletionsinHoxa11,Hoxc11,andHoxd11experiencedgreaterdefectsinhindlimblongbone

formation(Willik,2003).ThissuggeststhatHoxc11hasaneffectonhindlimbdevelopment.

Insituhybridizationrevealedthatcartilaginousmaterialonthegrowthplatesofthe

radius,ulna,and2ndthrough5thdigitsindicateHoxd11expression.ThissuggeststhatHoxd11

expressionisrelatedtogrowthplatepatterninginthedistalforelimb(Reno,2008).Chondrocytes

begintoorganizeintocolumnarandhypertrophiczonesbyday9andaperichondrialringforms,

bothindicativeofgrowthplateformation.Insituhybridizationatembryonicday15.5revealed

Hoxd11expressionaroundthepisiform.TheobservationsofcellpatterningandHoxd11

expressionprovideevidenceforgrowthplateformationinthemousepisiform(Kjosness,2014).

Potentialgrowthplateformationofthecalcaneushasnotbeenstudiedindepth,andthe

effectsofHoxa11,Hoxd11,andHoxc11oncalcaneusdevelopmentarelargelyunknown.While

wildtypemicedevelopedwelldefinedzonesbetweencarpalsandtarsalsearlyindevelopment,

Hox11mutantmicefailedtodevelopthesecleardivisions.MutationinHoxa11,Hoxc11,and

Hoxd11resultedinlessdefinitezonesinwristbones(carpals),providingevidencefortheeffect

ofthesegenesonproximalautopodformation.Triplemutantsalsofailedtodevelopdistinctive

calcaneusandtalusbones.Thezonesbetweeneachregionwereilldefined,preventingformation

ofarticularsurfacesbetweenthebones(Koyama,2010).

Hoxgeneinfluenceondevelopmentisindirect.Theregulatoryfactorsinvolvedinfluence

geneexpression,resultinginchangesincellactivityandinteractions.Studiesarecurrentlybeing

conductedoncandidategenes,genesthatpotentiallyhaveaninfluenceonpisiformand

calcaneusformation.Inthesestudies,theeffectsofmutantformsofthesegenesontheformation

ofthesebonesarebeingobserved.TheexaminationofcalcaneusdevelopmentandHox
expressioncouldbeexpandeduponinthefuturebyexaminingexpressionofthesecandidate

genesinrelationtoageandstageofdevelopment.Continuedstudiesonthesegeneswould

providemoreinformationonthemechanismsthatunderliebonedevelopment.

AimsofResearch

Whatpatternscharacterizecalcaneusformation?Howdothesepatternsandtheirtiming

correspondtoHoxexpressionpatterns?HowdothetimingofossificationandexpressionofHox

comparebetweenthepisiformandthecalcaneus?Thisresearchprojectiscomposedofthree

maincomponents.Together,theystrivetoadvanceunderstandingofdevelopment,thefactors

thatinfluencedevelopmentalprocesses,andtheevolutionaryimplicationsoftheseprocesses.

Aim1:Thefirstgoalofthisprojectistoobservecalcaneusformationinthemousehindlimb.

Theformationofthesecondaryossificationcenterandthesubsequentcellproliferationand

ossificationwillbeanalyzedintermsoftimingandpatterningatvariousstagesindevelopment.

Aim2:ThesecondgoalofthisprojectistoanalyzeHoxgeneexpressioninthecalcaneusduring

development.Thetimingandpatterningintheseprocessesshouldberelatedtoexpressionof

regulatoryfactors.Hoxa11andHoxd11expression,aswellasHoxc11,aresuspectedto

influencedevelopmentalprocessesinthecalcaneusandshouldbeobservedinthisanalysis.

Geneexpressionatdifferentlocationsinthegrowthplatewillalsoberecorded.

Aim3:Thethirdgoalofthisprojectistocomparepatternsincalcaneusformationtopisiform

formation.Thepisiformandcalcaneusareunusualshortbonesinthattheybothformgrowth

plates.Thedevelopmentalprocessesthatformthesebonessharesomeunderlyingmechanisms,

butthedetailsofthesemechanismsandextenttowhichtheseprocessesarerelatedareunknown.

Thiscomparisonshouldleadtoagreaterunderstandingoftherelationshipbetweenthesebones

and,throughfurtherresearch,themechanismsthataffecttheirmodificationinthehuman

lineage.

Methods

Micefrombirthto8weekshavebeencollected,stained,andpreservedbyRenoLab.The

contrastbetweenthealizarinredstainingofboneandalcianbluestainingofcartilageinthese

fullskeletonsamplesallowedfortimingofformationofcartilageandbonetobeexamined.In

viewingthesesamplesunderareflectinglightmicroscope,wefoundthattheappearanceofthe

secondarycenterbeginsaroundday9andthecalcaneusseemstobecompletelyfusedbythe

sixthweek.Thisinformationmayalreadybecomparedtothetimingofpisiformformation

observedbyKjosness,etal.inwhichthesecondarycenterbeganformationatday7andfusion

wascompletebyweek5(2014).Theseagesprovidetherangeoftimeinwhichgrowthplate

formationandbonegrowthoccur.

Sampleswithinthisobservedrangewillbeobtained,andhindlimbsfromthese

individualswillbeembeddedandsectioned.Thesesectionswillbeplacedonslidesandstained

withSafraninOandFastGreenstainstobeviewedthroughatransmittinglightmicroscope.

SafraninObindstochondroitinsulphateorkeratansulphate,polymersfoundincartilage,but

doesnotbindtocollagenfoundinbonetissue(Rosenberg,1971).FastGreenindicationof

ossifiedregionswillmarkacleardivisionbetweencartilageandbone(Reno,2006).This

differentialstainingwillallowforossificationandtheformationofthegrowthplatetobe

visualized.

Thedetectionofgeneexpressionispossiblethroughinsituhybridizationtechniques.

GeneexpressioncanbetrackedthroughthetranscriptionofgenesintoRNAandthepresence

RNAmoleculesmarkedwithvisualmarkers.Thisallowscellsexpressingaparticulargeneor

morethanonegenetobeviewedunderamicroscope(Mukai,1996).Probesaredesignedto
matchwiththetargetRNAsequenceaswellasmarkedwithahaptenthatwillallowtheprobeto

beviewed.Standardhybridizationprocedureswillbefollowedtoallowprobepenetrationinto

tissueandbindingtoRNAmolecules(Acloque,2008).Thesetechniqueswillbeimplementedon

mousesamplesofvariousageswithinembryonicdevelopmentfordirectcomparisonwith

pisiformdataandthelocationoftheprobesobserved.Inthiswaydifferentialgeneexpression

canbevisualizedthroughoutdevelopment.

Thisexperimentreliesonacomparisontopisiformdevelopmentinadditiontoobserving

developmentofthecalcaneus.Similarexperimentshavebeenconducted(Kjosness,2014)and

areinprogressinRenoLabonthemousepisiformwhichwillserveasthecomparisonforthis

experiment.Alargeamountofworkhasbeen(andcontinuestobe)doneonthepisiformwithin

thesamefacilitieswherethisresearchwillbeconducted,suggestingthatcomparisonwillbe

easilyaccomplished.Suchacomparisoninboneformation,especiallyinrelationtounderlying

mechanismsandevolutionaryimplications,isnecessaryfortheadvancementofbothstudies.

BroaderImplicationsandExpansion

Understandingthelinkbetweengenotypeandphenotypeisnecessaryforthestudyof

evolutionandtheeffectsandlimitationsofselectivefactors(Nedelcu,2004).Thisispossible

throughthestudyofdevelopmentalprocesses.Thisprojectwillexaminethedevelopmentofthe

calcaneus,abonethatisunusualinitsformationofagrowthplateaswellasthelargeeffect

environmentalfactorsplayonitsmorphology.ExaminationofcalcaneusdevelopmentandHox

geneexpressionwillaidinilluminatingtheprocessofgrowthplateformationandtheeffectof

involvedmechanisms.

Whilethehumanlineagehasseenareductioninpisiformlength(Kjosness,2014)

humansstillretaincalcaneusgrowthplateformation.Comparisonofthedevelopmental

processesofthesebonesmayrevealsimilareffectingmechanisms.Thisprojectalsoservesasa

startingpointfortheexaminationofmanygeneexpressionpatternsandtheirrelationshipstothe

growthplateformationandossificationprocess.Observationofhuman,ape,andmousepisiform

formationrevealedreductionandgrowthplatelossinthehumanlineage.Aswiththepisiform,

interspeciescomparisonswouldallowfortheinfluenceofevolutiononcalcaneusformationand

itscorrespondingeffectontheunderlyingmechanismstoberevealed.

References

AcloqueH,WilkinsonDG,andNietoMA(2008)Insituhybridizationanalysisofchickembryos

inwholemountandtissuesections.AvianEmbryology:2ndEdition.87:169185.

DavisAP,etal.(1995)Absenceofradiusandulnainmicelackinghoxa11andhoxd11.

Nature.

375:791795.

DeSilvaJM(2009)Functionalmorphologyoftheankleandthelikelihoodofclimbinginearly

hominins.ProceedingsoftheNationalAcademyofSciences.106(16):65676572.

FromentalRamainC,etal.(1996)Hoxa13andHoxd13playacrucialroleinthepatterningof

thelimbautopod.Development.122:29973011.

GilbertSF(2014)DevelopmentoftheTetrapodLimb.DevelopmentalBiology:10thEdition.

506507.

HallgrimssonB,WillmoreK,andHallBK(2002)Canalization,developmentalstability,and

morphologicalintegrationinprimatelimbs.AmericanJournalofPhysicalAnthropology.

35:13158.

Kjosness,KMetal.(2014)Thepisiformgrowthplateislostinhumansandsupportsarolefor

Hoxingrowthplateformation.JournalofAnatomy.

KoyamaE,etal.(2010)Hox11paralogousgenesarerequiredforformationofwristandankle

jointsandarticularsurfaceorganization.AnnalsoftheNewYorkAcademyofSciences.

1192:307316.

KronenbergHM(2003)Developmentalregulationofthegrowthplate.Nature.423:332336.

LatimerB,andLovejoyCO(1989)TheCalcaneusofAustralopithecusafarensisandIts
 ImplicationfortheEvolutionofBipedality.AmericanJournalofPhysicalAnthropology.

78:369386.

LovejoyCO,etal.(2003)DevelopmentalBiologyandHumanEvolution.AnnualReviewof

Anthropology.32:85109.

MukaiY(1996)InSituHybridization.PlantChromosomes:LaboratoryMethods.

NedelcuAM,andMichodRE(2004)Evolvability,Modularity,andIndividualityduringthe

TransitiontoMulticellularityinVolvocaleanGreenAlgae.ModularityinDevelopment

andEvolution.21:466.

RenoPL,etal.(2006)Ossificationofthemousemetatarsal:differentiationandproliferationin

thepresence/absenceofadefinedgrowthplate.TheAnatomicalRecordPartA:

DiscoveriesinMolecular,Cellular,andEvolutionaryBiology.288A(1):104118.

RenoPL,etal.(2008)Patternsofcorrelationandcovariationofanthropoiddistalforelimb

segmentscorrespondtoHoxdexpressionterritories.JournalofExperimentalZoology

PartB:MolecularandDevelopmentalEvolution.310B:240258.

Rosenberg,L(1971)ChemicalBasisfortheHistologicalUseofSafraninOintheStudyof

ArticularCartilage.TheJournalofBoneandJointSurgery.53(1):6982.

SkedrosJG,HuntKJ,andBloebaumRD(2004)RelationshipsofLoadingHistoryand

StructuralandMaterialCharacteristicsofBone:DevelopmentoftheMuleDeer

Calcaneus.JournalofMorphology.259(3):281307.

WellikDM,andCapecchiMR(2003)Hox10andHox11genesarerequiredtogloballypattern

themammalianskeleton.Science.301:363367.