Cell senescence contributes to tissue regeneration in zebrafish

Aging Cell. 2020;19:e13052.   | 1 of 5 https://doi.org/10.1111/acel.13052 wileyonlinelibrary.com/journal/acel Received:4March2019 | Revised:22July2019 | Accepted:23September2019 DOI: 10.1111/acel.13052 SHORT TAKE Cell senescence contributes to tissue regeneration in zebrafish Sabela Da Silva‐Álvarez1 | Jorge Guerra‐Varela2,3 | Daniel Sobrido‐Cameán4 | Ana Quelle2 | Antón Barreiro‐Iglesias4 | Laura Sánchez2 | Manuel Collado1 ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium, provided the original work is properly cited. ©2019TheAuthors.Aging CellpublishedbytheAnatomicalSocietyandJohnWiley&SonsLtd. 1LaboratoriodeCélulasMadreenCáncery Envejecimiento,InstitutodeInvestigación SanitariadeSantiagodeCompostela (IDIS),XerenciadeXestiónIntegradade Santiago(XXIS/SERGAS),Santiagode Compostela,Spain 2DepartamentodeZoología,Genética yAntropologíaFísica,Facultadde Veterinaria,UniversidadedeSantiagode Compostela,Lugo,Spain 3GeneaquaS.L,Lugo,Spain 4DepartmentofFunctionalBiology,Facultyof Biology,CIBUS,UniversidadedeSantiagode Compostela,SantiagodeCompostela,Spain Correspondence ManuelCollado,LaboratoriodeCélulas MadreenCánceryEnvejecimiento,Instituto deInvestigaciónSanitariadeSantiagode Compostela(IDIS),XerenciadeXestión IntegradadeSantiago(XXIS/SERGAS), SantiagodeCompostela,Spain. Email: [email protected] LauraSánchez,DepartamentodeZoología, GenéticayAntropologíaFísica,Facultadde Veterinaria,UniversidadedeSantiagode Compostela,Lugo,Spain. Email: lauraelena.sanche[email protected] AntónBarreiro‐Iglesias,Departmentof FunctionalBiology,FacultyofBiology, CIBUS,UniversidadedeSantiagode Compostela,15782SantiagodeCompostela, Spain. Email: [email protected] Funding information FundingatthelaboratoryofM.C.is providedbytheMinisteriodeCiencia, InnovaciónyUniversidades,Fondos EuropeosdeDesarrolloRegional(FEDER) (RTI2018‐095818‐B‐100).Workinthe laboratoryofA.B.‐I.wasfundedbygrants fromtheXuntadeGalicia(2016‐PG008) andthecrowdfundingplatformPrecipita (FECYT;2017‐CP081).ThelaboratoryofL.S. issupportedbytheRegionalGovernment XuntadeGalicia(ED431C2018/28). Abstract Cellular senescence is a stress response that limits the proliferation of damaged cells by establishing a permanent cell cycle arrest. Different stimuli can trigger senescence but excessive production or impaired clearance of these cells can lead to their accumulation during aging with deleterious effects. Despite this potential negative side of cellsenescence,itsphysiologicalroleasapro‐regenerativeandmorphogeneticforce has emerged recently after the identification of programmed cell senescence during embryogenesisandduringwoundhealingandlimbregeneration.Here,weexplored theconservationoftissueinjury‐inducedsenescenceinamodelofcomplexregeneration,thezebrafish.Finamputationinadultfishledtotheappearanceofsenescent cellsatthesiteofdamage,andtheirremovalimpairedtissueregeneration.Despite manyconceptualsimilarities,thistissuerepairresponseisdifferentfromdevelopmental senescence. Our results lend support to the notion that cell senescence is a positive response promoting tissue repair and homeostasis. KEYWORDS cellularsenescence,regeneration,tissueinjury,zebrafish 2 of 5 | DA SILVA‐ÁLVAREZ Et AL. 1 | INTRODUCTION, RESULTS, DISCUSSION Cellular senescence is a terminal cell response consisting on the implementationofapermanentcellcyclearrestandtheacquisitionofasecretoryphenotypewithcell‐to‐cellcommunicationproperties(Collado, Blasco,&Serrano,2007;Muñoz‐Espín&Serrano,2014).Exhaustion oftheproliferativecapacityofthecellleadstosenescence,andthe accumulation of these damaged cells in tissues from old individuals is consideredakeyelementintheprocessofaging(vanDeursen,2014). Despitethisdetrimentaleffect,thesenescenceresponsehasabeneficial side protecting damaged cells from proliferating. This is consideredthebasisofitstumor‐suppressivefunction(Colladoetal.,2007; Collado&Serrano,2010).Therecentidentificationofdevelopmentally programmed cell senescence during embryogenesis expanded ourviewofthepositiveactivitiesofthisresponse(Muñoz‐Espínetal., 2013;Storeretal.,2013).Senescenceduringdevelopmentpromotes cellturnover,tissueremodeling,and,paradoxically,growth.Asimilar positivepro‐morphogeneticactivityforcellsenescencehasbeensuggestedtooperateduringskinwoundhealinginmice(Demariaetal., 2014)andduringlimbregenerationinsalamanders(Yun,Davaapil,& Brockes,2015).Senescentcellsseemtoappearatwoundsitesafter injurytohelppromoteoptimalwoundhealing(Yun,2018). Here,wedecidedtoevaluatethesenescenceresponseinthe context of tissue injury using an animal model of complex tissue regeneration,thezebrafish.Tostudysenescenceaftertissuedamage, weamputatedthepectoralfinofadultfish(around1yearold)atapproximately 50% of its length and followed regeneration with time (Figure1a).Westainedfinsforsenescence‐associatedbeta‐galac‐ tosidase(SAbetaGal),themostwidelyusedmarkerofsenescence FIGURE 1 Pectoralfinamputation induces features of cell senescence. (a)Schematicrepresentationofthefin amputation system used throughout thestudy.(b)Representative photomicrographs of fins stained for SAbetaGalorphospho‐histone3(P‐H3, rightpanel)afteramputations(NA: nonamputated;8,16,and30dpa:days postamputation).Co‐stainingofP‐H3 wasdoneat8dpa.Arrowheadshows theamputationplane.(c)Schematic representation showing the different types of samples used in the study (NA:nonamputated;A:amputated; DIS:distalarea;PROX:proximal).(d) SAbetaGalactivitymeasuredusing Galactonsubstrateafter8,16,and 30dayspostamputation(dpa)(from5–10 animalspercondition).(e)Expression levelsbyQPCRofcdkn1a(leftpanel)and cdkn2ab(rightpanel)genesrelativetothe housekeeping gene rps11after8,16,and 30dayspostamputation(dpa).Results are presented as mean ± SD ***p<.001, **p<.01,*p<.05,n.s.nonsignificant | 3 of 5 DA SILVA‐ÁLVAREZ Et AL. (Dimri et al., 1995),after 8,16,or 30days postamputation (dpa), a time point in which fins were completely regenerated. Control stainings were performed on the contralateral unamputated fin or immediately after amputation to discard artifacts derived from unspecificstainingofdamagedtissue.Finsat8dpashowedintense bluestainingcomparedwithlightblueat16dpaandcompletelyabsentstainingat30dpa(Figure1b).Immunohistochemicalco‐staining withphospho‐histone3(P‐H3),amarkerofproliferation,at8dpa confirmedthattheSAbetaGal‐positivecellswerenotproliferating (Figure 1b).Tofurther confirmtheseresults,we used an alternativesenescencedetectionmethodmoreamenableforquantification, utilizing Galacton, a chemiluminescent substrate (Bassaneze, Miyakawa,&Krieger,2008).Wecollectedamputatedandnonamputated fins at different times during regeneration and split the amputatedfinsintoproximal(closertothebody)anddistal(theregeneratedarea)parts(Figure1c).Again,weobservedthat8dpawas thetimepointthatproducedastrongerSAbetaGalreactionandthis activitywasrestrictedtothedistalpartofthefin,theareawhere regenerationtakesplace(Figure1d).Incontrast,theproximalarea ofthe8dpafinandthedistalorproximalareasof16and30dpafins weremostlynegative(Figure1d). WealsoextractedRNAfromamputateddistalandproximalfins andunamputatedfins,tocheckfortheexpressionofsomegenes that have been linked to the induction of senescence in different species (Collado & Serrano, 2006; Hernandez‐Segura, Nehme, &Demaria,2018)andinzebrafish(Donninietal.,2010;Xiaetal., 2014).SimilartoourresultswiththeSAbetaGaldetection,thedistal partof8dpafinsshowedhigherexpressionlevelsofcdkn1a and cdkn2ab than the proximal part of amputated fins or the unamputated contralateral fin (Figure 1e). The expression of these senescence markersreturnedtonormallevelsafter16and30dpa,inlinewith ourobservationsusingSAbetaGal. Insummary,theseresultssupportthenotionofatransientinduction of cell senescence during fin regeneration as judged by increasedSAbetaGalactivityandupregulationoftheexpressionof key senescence genes such as cdkn1a and cdkn2ab.Asimilartransient induction of senescence has been previously reported during zebrafishheartinjuryandregeneration(Bednareketal.,2015). FIGURE 2 Removal of senescent cells impairsfinregeneration.(a)Schematic representation of the experimental strategy followed to analyze the effect of removing senescent cells from amputated finsafterincubationwithABT‐263for 48or72hr,ortreatedwithvehicle (VEH).(b)SAbetaGalactivitymeasured usingGalactonsubstrateat8days postamputation and after treatment with ABT‐263for48or72hr,orwithvehicle (VEH)(from5–10animalspercondition). (c)ExpressionlevelsbyQPCRofcdkn1a (leftpanel)andcdkn2ab(rightpanel)genes relative to the housekeeping gene rps11 at 8dpaandaftertreatmentwithABT‐263 for48or72hr,orwithvehicle(VEH). (d)Lengthofregenerate(%)reachedby amputatedfinsat8dayspostamputation andaftertreatmentwithABT‐263 relative to untreated amputated fins (five animalspergroup).(e)Representative photomicrographs of larval fins stained for SAbetaGalorp21,24hrafteramputation andcontrolfin(CTRL).Scalebars: SAbetaGAL:200µm;p21:75µm.Results are presented as mean ± SD ***p<.001, **p<.01,*p<.05,n.s.nonsignificant 4 of 5 | DA SILVA‐ÁLVAREZ Et AL. Triggering senescence after tissue injury could have positive or negative effects on the regenerative capacity of the damaged tissue,duetoitspotentialpro‐regenerativeandanti‐proliferativeactivities,respectively(He&Sharpless,2017).Todirectlyassessthe roleofsenescenceinductionduringfinamputation,wedecidedto induce the removal of these senescent cells from amputated fins. Forthis,wetreatedfishfor48or72hrwithABT‐263(Navitoclax),a senolyticcompoundthatbyinhibitingtheBcl‐2antiapoptoticfamily of proteins triggers specifically the death of the senescent cells (Changetal.,2016).WedeterminedtheactivityoftheSAbetaGal enzyme in extracts from unamputated fins as control and from the proximal and distal regions of amputated fins that were previously treatedwithABT‐263for48or72hrorthatwereincubatedwith vehicleasanegativecontrol(Figure2a).ABT‐263treatmentcaused areductioninSAbetaGalstainingandaconcomitantinductionof apoptosisintheregeneratingarea,asdeterminedbyTUNELstaining (FigureS1A–C).WequantifiedSAbetaGalactivityat8dpa,theday atwhichwehadobservedthepeakofsenescenceinduction.We confirmedtheinductionofSAbetaGalactivityinthevehicle‐treated fish and observed that the activity present in the extracts from the regenerating(distal)regionwasbluntedbytheABT‐263treatment (Figure2b).Furthermore,mRNAexpressionanalysisofcdkn1a and cdkn2abafterABT‐263treatmentalsoconfirmedthedrasticreduction in the levels of these senescence markers at the regenerating areaafter48and72hrofincubation(Figure2c). These results clearly show that it is possible to remove senescent cells from the regenerating area of injured fins by treating fish withthesenolyticcompoundABT‐263,sowewonderedwhatwas theeffectonregeneration.Forthis,wedeterminedtheregenerativecapacitybymeasuringthelengthofregenerateat8dpainfish treatedwithABT‐263for48or72hrorvehicle.Thisanalysisrevealedthatthe removalofsenescentcells byABT‐263treatment clearly impaired regeneration,withamputatedfins in fish treated with ABT‐263 showing a clear reduction in the length of regenerate compared with the one reached in vehicle‐treated animals (73.67%±14.92%and62.57%±12.87%after48or72hr,respectively)(FigureS1DandFigure2d).Apoptosishasbeenshowntobe acrucialprocessduringfinregenerationinzebrafish(Vriz,Reiter,& Galliot,2014).SinceABT‐263inhibitsproteinsoftheBcl‐2familyand thiscouldinterferewiththepro‐regenerativeapoptosisresponse, wedecidedtouseanalternativesenolytictreatment,quercetin(Zhu etal.,2015).Treatmentwithquercetinledtoasimilarreductionof SAbetaGalstainingandanimpairedregeneration(FigureS1E–G). The recent discovery of cell senescence during embryo development as part of a developmental program points to a role for senescence as a morphogenetic and proliferative force (Yun, 2018). Senescenceinductionduringadulttissueinjurycouldhaveresulted fromtheevolutionaryco‐optionofthisdevelopmentalprogramretained during adulthood. To further clarify the occurrence of senescence during development and tissue injury, wetested senescence induction in 3 dpf fish larvae after a complete spinal cord transection at the level of the anal pore which also damaged the surrounding body wall(musclesandskin).At2dayspostlesion(5dpfanimals),avery strongSAbetaGalstainingappearedintheskinandbody‐wallmuscles only at the injury site in lesioned animals and not in control unlesioned animals,orinportionsofthetrunkawayfromtheinjurysiteinlesionedanimals(FigureS2H).Thus,tissueinjury‐inducedsenescence isnotanexclusivepropertyoffinamputation,sinceadifferentkind of traumatic injury induces also cellular senescence in the skin and muscles of the trunk in zebrafish. Interestingly,andincontrasttolimbsinmice(Muñoz‐Espínetal., 2013;Storeretal.,2013),finsarenegativeforsenescencemarkers duringzebrafishdevelopment(Villiardetal.,2017).However,amputation of the caudal fin of 2 dpf larvae produced a clearly positive reactionforSAbetaGalactivityandp21expression(theproductof cdkn1agene)(Figure2e,andFigureS1I).Theseresultssuggestthat developmental senescence and tissue regenerative cell senescence are different cell responses triggered by different stimuli that might share some features such as their positive role promoting tissue remodelingandgrowth.However,ourdatadonotallowustodistinguish between a role in wound healing or during regeneration. Insummary,ourresultslendsupporttothenotionthattissue injury‐inducedsenescenceisapositiveresponsethatpromotesregeneration not only during mouse skin wound healing or salamander limbamputation,butalsoinzebrafish,awidelyusedanimalmodelof complex regeneration. ACKNOWLEDGMENTS WeacknowledgeMaríaOteroforexperttechnicalassistancewith histologicalanalysis.ABT‐263wasagenerousgiftfromAbbvie.M.C. isa“MiguelServetII”investigator(CPII16/00015). CONFLICT OF INTEREST Authorsdeclarenoconflictofinterest. AUTHOR CONTRIBUTIONS S.DS.‐A.performed andinterpretedmost ofthe experimentsand helpedwritingthemanuscript.J.G.‐V.,D.S.‐C.andA.Q.helpedwith experiments.A.B.‐I.,L.S.andM.C.designedtheexperiments,interpreted the results and wrote the manuscript. ORCID Manuel Collado https://orcid.org/0000‐0002‐0330‐0880 REFERENCES Bassaneze,V.,Miyakawa,A.A.,&Krieger,J.E.(2008).Aquantitative chemiluminescent method for studying replicative and stress‐in‐ duced premature senescence in cell cultures. Analytical Biochemistry, 372,198–203.https://doi.org/10.1016/j.ab.2007.08.016 Bednarek, D., González‐Rosa, J. M., Guzmán‐Martínez, G., Gutiérrez‐ Gutiérrez, Ó., Aguado, T., Sánchez‐Ferrer, C., … Flores, I. (2015). Telomerase is essential for zebrafish heart regeneration. Cell Reports, 12,1691–1703.https://doi.org/10.1016/j.celrep.2015.07.064 | 5 of 5 DA SILVA‐ÁLVAREZ Et AL. Chang,J.,Wang,Y.,Shao,L.,Laberge,R.‐M.,Demaria,M.,Campisi,J.,… Zhou,D.(2016).ClearanceofsenescentcellsbyABT263rejuvenates aged hematopoietic stem cells in mice. Nature Medicine,22,78–83. https://doi.org/10.1038/nm.4010 Collado, M., Blasco, M. A., & Serrano, M. (2007). Cellular senescence in cancer and aging. Cell,130,223–233.https://doi.org/10.1016/j. cell.2007.07.003 Collado,M.,&Serrano,M.(2006).Thepowerandthepromiseofoncogene‐inducedsenescencemarkers.Nature Reviews Cancer, 6, 472– 476.https://doi.org/10.1038/nrc1884 Collado, M., & Serrano, M. (2010). Senescence in tumours: Evidence from mice and humans. Nature Reviews Cancer,10,51–57.https://doi. org/10.1038/nrc2772 Demaria,M.,Ohtani,N.,Youssef,S.A.,Rodier,F.,Toussaint,W.,Mitchell, J.R.,…Campisi,J.(2014).AnessentialroleforsenescentcellsinoptimalwoundhealingthroughsecretionofPDGF‐AA.Developmental Cell,31,722–733.https://doi.org/10.1016/j.devcel.2014.11.012 Dimri,G. P., Lee, X.,Basile, G.,Acosta, M.,Scott, G., Roskelley, C., … Pereira‐Smith, O. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proceedings of the National Academy of Sciences of the United States of America, 92, 9363–9367.https://doi.org/10.1073/pnas.92.20.9363 Donnini,S.,Solito,R.,Cetti,E.,Corti,F.,Giachetti,A.,Carra,S.,…Ziche, M.(2010).Abetapeptidesacceleratethesenescenceofendothelial cellsinvitroandinvivo,impairingangiogenesis.The FASEB Journal, 24,2385–2395. He,S.,&Sharpless,N.E.(2017).Senescenceinhealthanddisease.Cell, 169,1000–1011.https://doi.org/10.1016/j.cell.2017.05.015 Hernandez‐Segura,A.,Nehme,J.,&Demaria,M.(2018).Hallmarksof cellular senescence. Trends in Cell Biology,28,436–453.https://doi. org/10.1016/j.tcb.2018.02.001 Muñoz‐Espín, D., Cañamero, M., Maraver, A., Gómez‐López, G., Contreras,J.,Murillo‐Cuesta,S.,…Serrano,M.(2013).Programmed cell senescence during mammalian embryonic development. Cell, 155,1104–1118.https://doi.org/10.1016/j.cell.2013.10.019 Muñoz‐Espín,D.,&Serrano,M.(2014).Cellularsenescence:Fromphysiology to pathology. Nature Reviews Molecular Cell Biology,15,482– 496.https://doi.org/10.1038/nrm3823 Storer,M.,Mas,A.,Robert‐Moreno,A.,Pecoraro,M.,Ortells,M.C.,Di Giacomo,V.,…Keyes,W.M.(2013).Senescenceisadevelopmental mechanism that contributes to embryonic growth and patterning. Cell,155,1119–1130.https://doi.org/10.1016/j.cell.2013.10.041 vanDeursen,J.M.(2014).Theroleofsenescentcellsinageing.Nature, 509,439–446.https://doi.org/10.1038/nature13193 Villiard,É.,Denis,J.‐F.,Hashemi,F.S.,Igelmann,S.,Ferbeyre,G.,&Roy,S. (2017). Senescence gives insights into the morphogenetic evolution of anamniotes. Biology Open, 6, 891–896. https://doi.org/10.1242/ bio.025809 Vriz,S.,Reiter,S.,&Galliot,B.(2014).Celldeath:aprogramtoregenerate. Current Topics in Developmental Biolology,108,121–151.https:// doi:10.1016/B978‐0‐12‐391498‐9.00002‐4 Xia,G.,Xin,N.,Liu,W.,Yao,H.,Hou,Y.,&Qi,J.(2014).Inhibitoryeffectof Lycium barbarum polysaccharides on cell apoptosis and senescence is potentially mediated by the p53 signaling pathway. Molecular Medicine Reports,9,1237–1241.https://doi.org/10.3892/mmr.2014.1964 Yun,M.H.(2018).Cellularsenescenceintissuerepair:Everycloudhas a silver lining. International Journal of Developmental Biology,62,591– 604.https://doi.org/10.1387/ijdb.180081my Yun,M.H.,Davaapil,H.,&Brockes,J.P.(2015).Recurrentturnoverof senescent cells during regeneration of a complex structure. eLife,4. e05505.https://doi.org/10.7554/eLife.05505 Zhu,Y.I.,Tchkonia,T.,Pirtskhalava,T.,Gower,A.C.,Ding,H.,Giorgadze, N.,…Kirkland,J.L.(2015).TheAchilles'heelofsenescentcells:From transcriptome to senolytic drugs. Aging Cell,14,644–658.https:// doi.org/10.1111/acel.12344 SUPPORTING INFORMATION Additional supporting information may be found online in the SupportingInformationsectionattheendofthearticle. How to cite this article:DaSilva‐ÁlvarezS,Guerra‐VarelaJ, Sobrido‐CameánD,etal.Cellsenescencecontributesto tissue regeneration in zebrafish. Aging Cell. 2020;19:e13052. https ://doi.org/10.1111/acel.13052