Inheritedcongenitalanteriorsegmentdysgenesis(ASD)disordersareamajorcauseofvision impairmentandblindness.Theyincludedevelopmentaloculardeficienciesoftheiris,corneaandlens inadditiontosignificantpredisposition,~50%,toglaucoma.Fromvariousstudiesinrecentyearsit hasbecomeclearthatmanycongenitaldisordersandpredispositionsarisefromearlydevelopmental defects,bothmajorandsubtle.Examiningthedevelopmentalprocessgivingrisetotheseessential componentsofthevisualsystemisthereforeafruitfulapproachtostudyingthedisease.Theanterior segmentderiveslargelyfromtheearlymigratingneuralcrestcellsthattargetspecificallytothe anteriorofthedevelopingretinaandarelooselyclassifiedasperiocularmesenchyme(POM).POM deficiencyhasbeendemonstratedincertaincasesofASD.Ibelieveamajorroadblockineffortsto treatandunderstandASDhasbeenthelimitedexaminationofPOMbiologyandfunctionduringearly oculardevelopment.Thishamperseffortstocharacterizetheetiologyofdiseaseandscreeningin ASDfamilies,inadditiontodelayingattemptsatiPSC-mediatedapproachesfortreatmentofASD.To circumventtheseproblems,weaimtocomprehensivelycharacterizePOMcellspecification, migration,targeting,differentiation,andultimatelycontributiontoanteriorsegmentdysgenesis.Thisis thenextlogicalandnecessarystepinorderforthefieldtocontinuetheevolutionoftherapyand screeningforglaucomaandASDdisorders.OurimmediategoalistotestthehypothesisthatPOM cellsdelineateintovarioussubpopulationsandspecifytobecometheanteriorsegment mesenchyme(ASM).Totestourhypothesisweproposetoemploythehighlyversatilezebrafish embryomodelsystemandcomprehensivelyexaminePOMcelldevelopmentwhilecatalogingtheir contributiontotheanteriorsegmentthroughgenetic,molecularandcuttingedgeinvivoimaging approaches.
Our specificaims are:
Aim1 :Characterizespecificationanddefinethecompositionof theASMtheusingacombinationofgeneexpressionanalysispairedwithlight-sheetinvivotime lapseimagingandconfocalmicroscopy.
Aim2 :Trackthemigratorybehaviorandlineagetrace distinctpopulationsofASMduringanteriorsegmentformationusinglight-sheetinvivotimelapse imagingandconfocalmicroscopypairedwithanovelfatemappingoptogenetictracingmechanism.
Aim3 :GenerateacomprehensiveASMtranscriptomeforscreeningnoveltargetsinASDpatient samplesusingacombinationofflowcytometrycoupledtoRNAseqandnextgenerationexome sequencing.
/RELEVANCETOHUMANHEALTH: Anteriorsegmentdysgenesis(ASD)disordershavedevelopmentalunderpinningsthatarecrucialto ourcomprehensionofvision,diseaseandadvancementoftherapy.Clinicalpracticewilldirectly benefitfromstudiesofanimalmodelslikezebrafish,whichprovidestremendousresearch advantagesincludingexuterodevelopment,opticaltransparencythroughoutdevelopmentand capacityforgenomeengineeringanddiseasemodeling.Thegoalofourproposalistogeneratethe firstcomprehensiveclassificationofperiocularmesenchymecellsinordertofacilitateeffortsin screeningforASDpredispositionandenablefutureinvivostemcelltherapy.
