Introduction
Approximately3millionpeopleovertheageof40sufferfromglaucoma,ablindingdisease.By2050,thatnumberwillriseto6million.1 Thisdisorder,alongwithage-relatedmaculardegenerationandretinitispigmentosa,affectthemostimportantpartoftheeye,theretina.Presently,therearenoknowncuresforthesediseases.2
Theretinapossessesasimilarstructureinallvertebrates,althoughcoldbloodedvertebratesareabletoregeneratetheretinawhereaswarmbloodedmammalssuchashumanscannot.3 Understandinghowthisprocessoccurscouldleadtocuresforretinaldegenerativediseases.Zebrafishprovideavaluablemodeltostudythesediseasesduetoseveralfactorssuchaslargeclutchsize,transparencyforthefirstfewdaysoflife,andquickdevelopment.4
Zebrafishretinalproliferationoccursattwolocations.TheCMZisthesiteofongoingproliferationduringdevelopmentandthroughadulthood.5 TheINL,however,containsspecializedcellsknownasMüllerglia.Thesecellsarethoughttode-differentiateandactasneuralprogenitorstoreplaceallofthecelltypesoftheretinaafterinjury(seefigure1A).5 Bystudyinglarvalzebrafishregeneration,cellsmaybeobservedastheymigrateanddifferentiate.Inordertostudyrepairresponsesusinglarvalzebrafish,itmustbeconfirmedthatthemechanismforregenerationisthesameasobservedinadultfish.
ExperimentalQuestionsWhendoretinalcellsrespondtoaninjuryinlarvalzebrafish?Whichcelltypesareinducedtoproliferatefollowinginjury?
Methods&MaterialsZebrafishPreparation• Tuba1a:GFPtransgeniczebrafishwerebredwithWTzebrafishforstudy.• At3dpf thefishwereanaesthetizedbybeingplacedinatricaine solution.(500mg/L)• Asmallprobewasusedtopiercethetoppartofthefish’srighteye.• Fishwerethentransferredtoa24-wellplatecontaininga0.5xpenicillinand
streptomycinsolution.Immunohistochemistry-• Zebrafishwerefixedat6,24,and48hpl in4%paraformaldehydeandcryosectioned• IncubatedwithblockingbufferPBS-T,10%BSA,and1%normaldonkeyserum• 0dpl tissuesectionswerelabeledusingRabbitanti-PH3primaryantibodies.• 1dpl and2dpl tissuesectionswerelabeledusingRatanti-BrdU andRabbitanti-GFP
primaryantibodies.• TissueswerelabeledwithDonkeyanti-RabbitCy3,Donkeyanti-RabbitAF488,and
Donkeyanti-RatCy3secondaryantibodiesandDAPI.ImagingandAnalysis• Imagingwasperformedusingafluorescencemicroscope(NikonMicrophot FXA,
Tokyo,Japan),equippedwithaRetiga 2000RdigitalcameraandZeissLSM700Confocalmicroscope.
• ImageswerecapturedusingQCapture software,colorizedwithPhotoshop,andanalyzedusingImageJ
• DatawasgraphedusingANOVAandSidak’s Multiplecomparisonstest
Conclusions• ProliferationintheINLincreasesfromtime
ofinjuryuntilatleast2dpl.
• Müllergliaactivationbeginsat2dpl.
• ProliferationoftheMüllergliaisminimalat1dpl.At2dplmostMüllergliaproliferate.
• Moreproliferationoccursinthelesionedeyesthanthelefteyesat2dpl.
• Themechanismforretinalregenerationappearstobethesameinbothadultandlarvalfish
FutureDirections• Useofliveimagingonlarvalzebrafish
wouldbebeneficialtoseemigratingcellsandneuralprogenitors.
• Studyprogenyoftheneuralprogenitorsandmigrationofthosecells
• Useofalargersamplesizeforquantification purposes
References1(LearnAboutGlaucoma.(n.d.).RetrievedJuly20,2017,fromhttps://nei.nih.gov/nehep/programs/glaucoma/learn-about))2FactsAboutAge-RelatedMacularDegeneration.(2015,September01).RetrievedJuly21,2017,fromhttps://nei.nih.gov/health/maculardegen/armd_facts3Than-Trong,E.,&Bally-Cuif,L.(2015)..Glia, 63(8),1406-1428.doi:10.1002/glia.228564Kimmel,C.B.,Ballard,W.W.,Kimmel,S.R.,Ullmann,B.,&Schilling,T.F.(1995). DevelopmentalDynamics,203(3),253-310.doi:10.1002/aja.10020303025Wilson,S.G.,Wen,W.,Pillai-Kastoori,L.,&Morris,A.C.(2016). ExperimentalEyeResearch, 145,75-87.doi:10.1016/j.exer.2015.11.002
RetinalRegenerationinLarvalZebrafishNoahWille1,AlexConyers2,AlisonNevin3,ElizabethSandquist4,JeffreyEssner4,DonaldSakaguchi4
1NorthPolkHighSchool,2WaterlooWestHighSchool,3UniversityofNorthwestern-StPaul,4Genetics,Development,andCellBiology,IowaStateUniversity
Results
Fig.2At0dplthereisnoproliferationintheINLandnoactivatedMüllerglia.Theinjurysiteisvisibleat0dayspostlesionasmarkedbythearrow.Red=PH3,Blue=DAPI,Green=Tuba1.IHCperformedbyAnnaZmich.
Fig.3At1dplMüllergliaareactivatedwithverylittleproliferation.Red=BrdU,Blue=DAPI,Green=Tuba1
Fig.4At2dplmanyMüllergliaareactivatedandproliferating.A)20Ximageoflesionedeye,B)40xconfocalimageofproliferatingMüllerglia.Red=BrdU,Blue=DAPI,Green=Tuba1
Discussion
Fig.5QuantificationofMüllerGliaactivationandlabelingforproliferation.GraphAshowsnosignificantdifferencebetweentheamountofGFP+cellsineithertimepointoreithereye.GraphBshowsnosignificantdifferencebetweentheamountofGFP+cellsthatareco-labeledwithBrdU between1dpland2dpl.GraphCshowsasignificantdifferencebetweenboththelesionedeyes(1dpland2dpl)andthelesionandcontroleyesofthe2dplfish.N=2-3
AcknowledgementsAbigthankyoutoAdahLeshem,JenniferLillo,andMaureenGriffinandanyonewhohelpedtoorganizetheYESprogram.ThankyoutotheMcGrail andEssner labsforprovidingtheZebrafishandAntibodiesusedinourlab.ThankstoAnnaZmich fortheimagesanddataforourposter.Thankstoallofthoseinthelabwhohelpedsupportmethroughoutmyexperience.ThankstomyparentsJasonandSuzanneforalwaysbeingtheretosupportme.Andfinally,thankstomyhighschoolChemistryandBiologyteachers;KimKult,andRosalieEimers,forgivingmetheexperiencetoparticipateintheYESprogram.
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Fig.1A)Imageofthelesionsiteoftheeyemarkedbyanarrow.B)Diagramofazebrafishretina.
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