navigating the bonebase webportal - kompbonebase.org/bonebase/new/navigate_db.pdf · navigating the...

NavigatingtheBonebaseWebportal

Geneticsisamajorcontributortosusceptibilityorresistancetoosteoporoticbonedisease.HumanGWASandmouseKOscreeningprogramsestimatebetween500and2000+genescaninfluenceadultbonemass.Thisrealityposesamajorbioinformaticschallengetoourfieldtoidentify,organizeandclassifybonephenotypinginformationinabigdataformatinawaythatismeaningfultotheskeletalbiologistandcanbeintegratedwithothergenetic,proteomicanddruginteractiondatabases.Thepresentationwilldescribeoureffortstodevelopanexperimentalandbioinformaticsplatformthatcouldbeafunctionalbeginningtowardsabigdatasolutionforgenesimpactingskeletalvariation.ItisbasedonourexperienceofscreeningmicefromtheInternationalMousePhenotypingConsortium(IMPC)inwhichindividualgeneshavebeeninactivatedwithinthesamegeneticbackground,andtheresultingknockoutlines(KOs)arecharacterizedatmultipletissuelevels.OurunselectingscreenofKOmiceisbasedonµCTofthedistalfemurandvertebra.ThebonesoftheKOlineswithamajorvariationintrabecularorcorticalbonevolumewerefurthercharacterizebyahistomorphometricanalysis.Howthesemethodswereemployed,whathasbeenlearnedtodateandhowtheskeletalbiologycommunitycanparticipateinthiseffortwillbediscussed.

ExperimentalDesign:Costvs.DepthofAnalysis;Consistencyinmicrobiomeandmousemanipulation. •HomozygousKO–WeexpandedbreedingpairsofviableKOlinesfromtheJacksonLaboratoryIMPCproductionfacility(intheUS,itiscalledtheKnockOutPhenotypingProject,KOMP).Weelectednottostudyheterozygotesofembryoniclethalorpoorbreedinglinesbecauseoftheextraexpenseofbreedingandgenotyping. •Controls–EachmonthasetofC57BL6/NJmiceweregeneratedforanalysisbythesameworkflowastheKOlines. •Controlledbreedingandharvesting-Theoffspringsofeachlinesweregrownto12weeksofage.Theywereinjectedwithcalcein7daysandalizarinecomplexone2dayspriortosacrifice.Allthebreeding,cagemanagementandtissueharvestingwasperformedinthesameroomattheJacksonLaboratory.TissuesampleswereshippedtoUCONNforanalysis.

Tissueanalysis:Controlledworkflow,observer-independentdataaccumulation.

•Samplecollectionanddistribution:EachKOorcontrollinewascollectedoverthecourseof1-4monthsofbreedingandgrowingdependingonthefecundityoftheline.Thecollectionwasconsideredcompleteafter8malesand8femaleswereobtained.Alaboratoryinformationmanagementsystem(LIMS)controlledtheflowbygeneratingbarcodesforeachsampleandeachhands-onstepintheprocess. •ScreeningbyµCT–Thedistalfemurand2ndlumbarvertebrawerescannedat16µresolutioninusingacarouselcontainingmulti-sampleholders.CustomsoftwaredeterminedtheROIofeachtissuefromwhichthearchitecturalfeaturesweredetermined.ThecalculatedoutputwasuploadedintotheLIMS,thedatawasinspectedbytheteamandtheinitialinterpretationwasreleasedfordistributiononthewebportal. •Bonehistomorphology–KOlineswithasignificantvariationbyµCTwereprocessedusingatape-transfercryohistologicalprotocoloptimizedfornon-decalcifiedtissues.Either4distalfemuror4lumbarvertebra(#4-6)wereembeddedtogetherinaparallelorientationsothatthetapecaptured4samplescollectedfrom3depthsseparatedby~50µ.Afullanalysisofafemurorvertebraiscollectedon6slideseachcontaining8sectionsat3sectiondepthsfromthemaleandfemaleKOline.Thesectionsareprocessedfor4roundsofstainingandautomatedimagingusingtheZeissAxioscan.(1)Accumulatedmineralandmineralizationlines.(2)DemineralizationandTRAPstainingusingtheElf97substrate.(3)Alkalinephosphatasestainingusingfastredsubstrate.(4)toluidineblue.Allstepsareaqueoustopreventtissueshrinkage. •Imageanalysisofhistologicalimages–The10grayscaleimagefilesthatareproducedbytheAxioscanareoverlayed,verticallyaligned,backgroundcorrectedandbinarized.Theregionofinterestisdeterminedandeachfluorescentsignalisprojectedtothesurfaceofthetrabecularbone.Theextentofeachfluorescentsignal,whichrepresentsaspecificbiologicalactivity,isexpressedasthepercentageoftrabecularsurface.Theprimarymeasurementsare:BV/TV,TbTh,MS/BS(%labelingsurfaces),MAR(distancebetweenmineralizinglines),AP/BS(%APpositivesurfaces)andTRAP/BS(%TRAPpositivesurfaces.TheanalysisallowstheAPandTRAPtobesubdividedintosignalsthatareoverlyingamineralizinglabelingsurface(LS)onnonlabelingbonesurface(NL).Thisisinterpretedtodistinguishactiveosteoblasts(AP/MBS)fromboneliningcells(AP/NBS),andremodeling(TRAP/MBS)andresorbing(TRAP/nMBS)bonesurface.ItalsoidentifiessiteswhereAP,TRAPandMBScoincide,whichis

interpretedasaboneremodelingunit.TheresultingcalculatedvaluesareuploadedintotheLIMS.

DataPresentation:Navigatingthewebportal,www.bonebase.org. Thewebsitehastwocomponents,staticanddynamic.OntheleftpanelarelinkstostaticpresentationsofthedetailsoftheµCTandhistologicalmethods,definitionsusedandhowtonavigatethedynamicdatabase(Fig1).MostimportantistheexplanationonhowcallsaremadethataKOhasameaningfulvariationinbonearchitectureorcellularcomposition.ItisfromthisinformationthatthedynamicdatabaseorganizesthedifferentphenotypesoftheKOmouselines.

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•Useofcontroldata–Themonthlysetofcontrolmice(37sets)providedtoopportunitytoobservetheconsistencyofµCTandhistologicalmeasuresovertime.ForµCT,itclearlydemonstratedthegreatervarianceinmalevsfemaleanimalsaswellasthemonth-to-monthchangesthatcannotbeexplainedbyseasonalconditions.Forhistomorphometry,thecontrolswereveryusefulforidentifyingerrorsinthehistologyandimageanalysisthatoncecorrectedsignificantlyimprovethevarianceofthemeasurements.AnumberofcontrolstepswereintroducedtoensurethattheenzymaticstainsforAPandTRAPremainedconstantacrosscontrolandKOlines.

•MakingabnormalcallsofKOlines–BasedonthesignificancetestusingtheIMPC-developedstatisticalpackage(Phenstat),theLIMSidentifiesmeasurementsthatareregardedasmeaningfultothebonebiologist.Forexample,aBV/TVoftrabecularboneofdistalfemurofatesttocontrolratio>1.35or<0.75isconsideredtobesignificant.ThesecriteriaplacethecalledKOlinebeyondthebodyofthedistributioncurveofthecontrolsandthemajorityoftheKOlines(fig2A).

•Searchpage–UponenteringthehyperlinkedKOMPExperimentalData(Figure1yellowarrow),thescreenpresentstherangeofskeletalandbodysizevariationofthe220linesthatwereanalyzed.Theselectionboxesthatarescoredaslow,normalorhighbasedontheµCTvaluesforBV/TVofthedistalfemurorvertebra,thetotalvolumeofthetrabecularspaceofthedistalfemurorvertebra,thetotalvolumeforthefemoralcortexandthebodyweight.KOsthatfitanyofthesemeasurementsaloneorincombinationwillbefound.Clickingthemaleorfemalelinkwillsortbasedonthetesttocontrolratiovalue.

•Detailpages–ClickingthegeneIdhyperlinkwillpresentasummarytablethatprovidesallofthecallmeasurementsforµCT,histomorphometryandbodycompositionas

reportedbytheIMPCwebportal.HyperlinktitlesovertheµCTandhistomorphometryleadtotablesthatprovidethemeangroupdataandindividualmousedata.Inadditionthetabsabovethetableprovidenewscreens:

(1)FrequencydistributiongraphoftheBV/TVmeasurementrelativetothecontrolandotherKOMPlines(figure2A);

(2)HorizontalslidergraphsoftheµCTandhistomorphometrytest/controldatainwhicharatioof1isplotedasunchanged(0.00)Onetabisforthefemurandtheotherforthevertebra(figure2B);

Figure2:A.FrequencydistributiongraphsofBV/TVthatarebinnedinto1%intervals.TheredarrowpointstoKO.B.Slidergraphsoftheratiooftestvscontrol-1oftheprimaryµCTmeasurementsoftrabecularbone.Valuestotherightof“0”arethefractionalincreaseordecreaseofthetestmeasurementversusthecontrol

(3a)LinkstoPubmed.ArticlesrelatedtotheKOrelatedgenethathavebeenselectedbyuscanbeviewed;(3b)Linkstosupportinginformation.ThispageprovideslinkstoothersitesincludingtheIMPCfindingsfor

theKOline,OMIN,GWAS,MGI,geneexpression(EMBLandVisigene)andtextassociations(iHOP);(4)Classificationandinterpretation(notcurrentlyactive).Thisisaworkinprogressinwhichthewebportal

staff,externalexpertsandregistereduserscanprovidetheirinterpretationofthephenotypeitsgenetic/molecular

Figure1:EntrancetoKOMPdatascreen.

basis.Threelevelsofclassificationarebeingdevelopedthatbinthephenotypesbasedonthearchitectural,cellularandmolecularfindings,againreflectingtheinterpretationoftheseclassificationobjectives.

SelectingaKOline–ScreeningbyµCTandbodyweightplacesKOindifferentgroupings.Thesearchscreenprovidesentryintospecificlinesforgreaterdetail(figure3).TheµCTdata,presentedas

theratiooftestvscontrol,canbeselectedbasedonthe4primarymeasures(BV/TVandtotalvolumeoftheROIofthefemurandvertebra,thecorticalvolumeoffemurandbodyweight)byclickingtherangeofvalues.Byselectonevalue,allKOlinesthatmeetthesinglecriteraareshown,whileselectingmultiplevaluespulluponlytheKOsthat

Figure3:ScreenshotoftheseachpageforselectingKOmiceforfurtheranalysis.Thecheckboxesunderthe4selectionoptionswillcallupthoseKOlinesthatmeetasingleor“and”combinatorialselection.meetallofthecriteria.Figure4showsthetop10resultsofaloworhighBV/TVonlyrequest.ThissearchisplacesalltheexaminedKOsinthesamecontextofgeneticbackgroundandexperimentaltechniqueallowingtherelativemagnitudeofoneKOtotheentirepopulationofKOstobeappreciated.

Figure4:Selectedscreenshotofthe10mostaffectshitsforeitherlow(A)orhigh(B)trabecularbonemass.Thenumberaretheratioofthetest/controlvaluesforµCTdeterminedBV/TV.Notshownaretheassociatedvaluesfortotalbonevolume,corticalvolumeandbodyweight.ThearrowsidentifyKOsthathavebepreviouslypublished.Theyellowandpurpleboxeshighlightthevaluethatwassortedbythesearchalgorithms.

Whenadditionalselectioncriteriaareapplied,groupsofKOswithsimilarfeaturesofbonesize(TVandcorticalvolume)andbodysize(weight)emerge.Figure5showsthelistofmicewithalowBV/TVbutotherwisewithanormalbonesizeandbodyweight.MostoftheseKOswerenotdetectedbytheIMPCscreenthatisbasedonwholebodyDXA.NotethattheabnormalBV/TVcanbeinthefemurorvertebraorboth,andcanbeobservedinfemalesormalesorboth.Presumably,thesemutationsaffecttrabecularbonedirectlywithouthavingwidespreadmetabolicorbasichousekeepingeffects.Incontrast,othergroupingcanbegeneratedinwhichlowBV/TVisaccompaniedbyalowbonesizeandorlowbodyweightorboth(Figure6).Somemembersofthiscategoryprobablyrepresentmutationsthathavemultiplemetabolicorbasichousekeepingfunctions.Anotherpossibilityisthatsomerepresentgenesthatcouldbeclassifiedasfrailtyassociatedgenes.Thegoalofthisarchitecturalclassificationistoprovidesomecontextthatwillassisttheviewertofocusongenesthatfittheirresearchinterest.

Figure5:ResultsofasearchofallKOlineswithalowBV/TVineitherthefemurorvertebraorbothandnothaveanabnormalmeasureofbonesizeorbodyweight.Tobeincludedinthelist,theBV/TVhastobelessthan0.75inthefemurand0.80inthevertebra.Notethatgenenumbers2,10,11,15and18aremalerestricted,while12,13,14and19arefemalerestricted.Alsothegenenumbers12,14,15arefemurrestrictedwhile17,19,20arevertebrarestricted.

Figure6:Classificationoflowtrabecularbonevolumeinassociationwithmeasuresofbonesizeandbodyweight.Asimilarclassificationcanbeconstructedbasedonhightrabecularbonevolume(HTBV).

ExaminingaKOlineindetail:Interferonregulatoryfactor-8(IRF-8),atranscriptionfactorexpressedinimmunecells,isakeyregulatorymoleculeforosteoclastogenesis.IRF-8expressioninosteoclastprecursorsisdown-regulatedduringtheinitialphaseofosteoclastdifferentiation.PreviouslypublishedKOmicehaveaverylowtrabecularbonemassduetoactivatedosteoclastlineage.ItisthemostsevereLTBVKOthatwehaveencountered.Toexaminethedetailsofouranalysis,clickthegeneabbreviationonthesearchpageandthefrequencydistributiongraphsoftheBV/TVinthefemurandvertebrainbothsexesareshown(figure7).ThearrowpointstotheIrf8valuestoshowitspositionrelativetotherunningcontrolsandtheotherKOlines.

Figure7:CompositeimageofIrf8valuesshowfromthesearchpageandthefrequencydistributiongraphsthatplacethevaluesofIrf8relativetothemonthlycontrolsandthestudiedKOlines.ThearrowisaddedtoemphasizetheIrf8values.

Thetabsatthetopofthefrequencydistributiongraphwillpresentadditionalinformationontheline.ClickingthesummarytabbringsthesummarytableinwhichspecificmeasurementsinµCT,histomorphometryandbodycompositionarecallasbeingeitherlow,normalorhigh(figure8).Theheaderforeachtablesectionisahyperlinktotheprimarygroupandindividualmousedata.Inthecaseofhistomorphometry,athirdlevelofdetailisprovidedinwhichthehistologicalimagesandcalculatedmeasurementforthethreesectionsfromeachbonesamplecanbeviewed.

Themeasurementsthatsupportthesecallsaregraphicallypresentedinseparatetabsforthefemurand

vertebra(figure9,femurslidergraph).TheAgroupingisµCTandhistologicalmeasuredoftrabecularbonewhiletheBgroupingisthebonesizeincludingTVoffemurandcorticalvolumeoffemurandthebodysizeasassessedbyweightandfemurlength.ThegraphsshowthatbothµCTandhistologyagreethatthelowBV/TVisprimarilyalossin

Figure8:Summarytable.A.DatafromµCTanalysisincludingBV/TVandTVoffemurandvertebra,andthecorticalbonecrosssectionalmeasuresandfemurlength.B.Datafromhistomorph-ometryforbothbonesthatincludesthe8categoricalmeasureofformation,

resorptionandremodeling.C.bodycompositionobtainedbyDXAduringtheIMPCphenotyping.Theweightatsacrificetabisameasurementmadebyourgroup.Thedatasupportingtheseoutcomecallsarehyperlinked.

trabecularnumber.Boneandbodysizeareinthenormalrange.PanelCpresentstheboneformingmeasuresfromthehistomorphology.Arelativelymodestincreaseinboneformationrate(BFR)primarilyduetoanincreaseinmineralizingsurfaceswasobtainedfromthedynamiclabelingstudy.Totalosteoblastsurfaceswasalsomoderatelyincreasedanditwasdistributedprimarilytoboneformingvsboneliningsurfaces.ThemostdramaticmeasurescomefromtheTRAPstudy(panelD).Totalosteoclastsurfacewasincreasedandagreaterproportionoftheactivitywaslocatedonboneforming(remodeling)ratherthaninactivesurfaces.HowevertheTRAPwasprimarilylocatedatsiteswheretheAPandmineralizationcoincidedwhichweregardasaremodelingunit.Thisisoneofthehighestvalueswehaveobservedtodate.Thesamefindingwasobservedinthevertebra.Theconclusionfromthehistomorphometrystudyisastateofhighboneturnoverthatislocalizedattheconfluenceofmatrix-formingosteoblasts.

Figure9.SlidergraphsofalltheµCTandhistomorphmetricmeasurementofthefemur(maleandfemale).Thehorizontalbarsreflectthefractionalincrease(rightside)ordecrease(leftside)ofthe0.00lineofeachmeasurement.Seetextfortheinterpretationofthedata. ThelinktabpresentstheresultsofsearchesofvariouswebsitesforinformationregardingthisKOline.The

Figure10:LinkpageshowingthevarioushyperlinkstoIMPC,Pubmed,OMIN,MGIandotherusefulsites.Seetext.

linktothehomepageandabnormalfindingpage(redarrow)isausefulstartingpointandtotalbodybonemineralcontent(BMC)obtainedbyDXA(greenarrow)canbeinformative.Otherlinksincludetheweightcurves,leanandfatmasscompositionsobtainedbyDXA.Adirectlinktopubmed(bluearrow)andpublicationthataredirectlyrelatedtotheKOlinecanbeviewed.FinallylinkstoRNAexpressiondatabases(magentaarrow)ortoOMINorGWAS(yellowarrow)providesinformationonthehumanrelatedinformation.

FuturePlansfortheWebPortal

Goingforward,someofthenewfeaturesthatwillbeaddedtoourphenotypingworkflowwillinclude:•BreedtheIMPCmiceinhouse–Byobtainingbreedersfromanexternalsourceandestablishingthe

breeding/growinglinesatthesamelocationthatthesamplesareprocessed,aconsistencyinthemicrobiome,housingandharvestingismaintained.ThisallowsobtainingtheIMPCmicefromdifferentproductionfacilitiesusingonesetofcontrolmiceratherthanacontrolforeachperipheralbreedingsite.ThiswillincreasethenumberandtypeofKOlinesthatwecanevaluate.Inaddition,itprovidesamechanismforanexternalinvestigatortopurchaseanIMPCKOmouseofinterest,haveitdeliveredtotheUCONNbreedingfacilityandhavetheoffspringsanalyzedinoursameworkflow.TheadvantagefortheexternalPIistheavoidanceofthecostforcontrolsetofanimalsbecausetheyarebeingproduceandanalyzedaspartofourongoingstudies.Formoredetailsseethecoststructureforthisbreedingandanalysisoption.

•RapididentificationofaKOwithanexceptionalarchitecturalphenotype-WewanttoextendthebreedingofKOlineswithamajorbone/bodyvarianceforproductionofadditionalanimals,embryocryopreservationandpotentiallyfortransfertoanexternalinvestigator.

•DeeperinvestigationofKOlineswithevidencesuggestiveofafrailtyorfitnessphenotype-TheIMPCusesDXAtomeasureleanandfatbodymasscomposition.TissuewillbetakenfromKOlineswithanabnormalDXAvalueforeithervaluetoassessskeletalmuscleforfibersizeandnumberandboneforµCTassessmentofbonemarrowfat.

•Classification–Theconceptofaclassificationsystemforthearchitecturalandbodycompositionmeasurementswasintroducedinfigures4and5.TheKOlineswereidentifiedmanuallybutinthefuturecanbeproducedasadefinedgroup.Thecriteriaforthegroupingswillneedtobedefined,andsubsequentlymodifiedasmoreKOareencountered.Asimilarclassificationisenvisionedforthehistomorphologicalfindings.ThiswillallowidentificationofKOlineswithadefinedcellularbasisforthearchitecturalphenotype,astepthatwillrequiredevelopingcriteriafordefinedgroupings.Finally,themolecularpathwaysthatinterconnectwiththeKOlineneedtobedevelopedtounderstandwhytheKOexertsitsarchitecturalandcellularphenotype.ClassificationbasedonpathwayneedstobedevelopedtoidentifydifferentKOlinesthatmaptosimilarpathways.

•ExternalInput–Oureventualgoalistomakethissiteevolveintoacommunityresourceforgenesthatimpactthemineralizedskeleton.Weneedtoelicitinputfromexpertsindifferentcellandmolecularaspectsofskeletalbiologytohelpdevelopmeaningfulclassificationsystemsandtoprovidetheirinterpretationoftheassembledinformation.Wehaveinvitedalimitednumberofexpertstobeginthisprocess,butwillextendtheinvitationtootherswhowishtoparticipate.Aregistereduserlistwillbedevelopedwhowillbesegmentedonspecificareaofexpertise/interest,e.g.osteoclastorosteoblastbiology,coupling,frailty,fitness,aspecificmolecularpathway.WhenanewKOlineisidentifiedthatmayfitoneoftheseinterestareas,theinterestedindividualswillbeinvitedtoavideoconferencetoinspectthedataandtocontributetotheinterpretation.RegistereduserscanrequestaKOlinethatiscurrentlyactiveandshowspreliminaryµCTdataofanabnormalarchitectural/somaticphenotype.Iftheyreceivethemice,theywillberequestedtosharetheresultsoftheirstudyonthewebsite.

•Useoftheworkflowfornon-IMPCmice–Becausetheworkflowishighlyautomated,wecanperformacomprehensiveµCTandhistomorphometricanalysisatasignificantcostandtimeadvantagefromtraditionalmethods.HoweverunliketheIMPCmice,eachinvestigator-derivedlinewillrequireitsownsetofcontrols.Amechanismforproducingandshippingthebonesamplesthatintegratewithourworkflowhasbeendeveloped,oraninvestigatorcansendthebreederstooursitetohavetheentireprocessperformedinhouse.Histomorphometrycoresthatwishtoestablishthemethodsintheirinstitutionandusetheimageanalysisplatformarewelcomedtovisitandhaveahands-onlearningexperience.Theseoptionsarepresentedelsewhere.