https://doi.org/10.4081/ejh.2024.3977
Senescence-associated ß-galactosidase staining over the lifespan differs in a short- and a long-lived fish species
Submitted: 25 January 2024
Accepted: 21 February 2024
Published: 29 February 2024
Accepted: 21 February 2024
Abstract Views: 587
PDF: 321
Supplementary: 21
HTML: 2
Supplementary: 21
HTML: 2
Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
Authors
During the aging process, cells can enter cellular senescence, a state in which cells leave the cell cycle but remain viable. This mechanism is thought to protect tissues from propagation of damaged cells and the number of senescent cells has been shown to increase with age. The speed of aging determines the lifespan of a species and it varies significantly in different species. To assess the progress of cellular senescence during lifetime, we performed a comparative longitudinal study using histochemical detection of the senescence-associated beta-galactosidase as senescence marker to map the staining patterns in organs of the long-lived zebrafish and the short-lived turquoise killifish using light- and electron microscopy. We compared age stages corresponding to human stages of newborn, childhood, adolescence, adult and old age. We found tissue-specific but conserved signal patterns with respect to organ distribution. However, we found dramatic differences in the onset of tissue staining. The stained zebrafish organs show little to no signal at newborn age followed by a gradual increase in signal intensity, whereas the organs of the short-lived killifish show an early onset of staining already at newborn stage, which remains conspicuous at all age stages. The most prominent signal was found in liver, intestine, kidney and heart, with the latter showing the most prominent interspecies divergence in onset of staining and in staining intensity. In addition, we found staining predominantly in epithelial cells, some of which are post-mitotic, such as the intestinal epithelial lining. We hypothesize that the association of the strong and early-onset signal pattern in the short-lived killifish is consistent with a protective mechanism in a fast growing species. Furthermore, we believe that staining in post-mitotic cells may play a role in maintaining tissue integrity, suggesting different roles for cellular senescence during life.
Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. Hallmarks of aging: An expanding universe. Cell 2023;186:243-78.
DOI: https://doi.org/10.1016/j.cell.2022.11.001
Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res 1961;25:585-621.
DOI: https://doi.org/10.1016/0014-4827(61)90192-6
Hernandez-Segura A, Nehme J, Demaria M. Hallmarks of cellular senescence. Trends Cell Biol 2018;28:436-53.
DOI: https://doi.org/10.1016/j.tcb.2018.02.001
Campisi J. Aging, cellular senescence, and cancer. Annu Rev Physiol 2013;75:685-705.
DOI: https://doi.org/10.1146/annurev-physiol-030212-183653
Childs BG, Durik M, Baker DJ, van Deursen JM. Cellular senescence in aging and age-related disease: From mechanisms to therapy. Nat Med 2015;21:1424-35.
DOI: https://doi.org/10.1038/nm.4000
He S, Sharpless NE. Senescence in health and disease. Cell 2017;169:1000-11.
DOI: https://doi.org/10.1016/j.cell.2017.05.015
Herranz N, Gil J. Mechanisms and functions of cellular senescence. J Clin Invest 2018;128:1238-46.
DOI: https://doi.org/10.1172/JCI95148
Kowald A, Passos JF, Kirkwood TBL. On the evolution of cellular senescence. Aging Cell 2020;19:e13270.
DOI: https://doi.org/10.1111/acel.13270
Roger L, Tomas F, Gire V. Mechanisms and regulation of cellular senescence. Int J Mol Sci 2021;22:13173.
DOI: https://doi.org/10.3390/ijms222313173
Schwartz RE, Conboy IM. Non-intrinsic, systemic mechanisms of cellular senescence. Cells 2023;12:2769.
DOI: https://doi.org/10.3390/cells12242769
Singh PP, Demmitt BA, Nath RD, Brunet A. The genetics of aging: A vertebrate perspective. Cell 2019;177:200-20.
DOI: https://doi.org/10.1016/j.cell.2019.02.038
Baker DJ, Childs BG, Durik M, Wijers ME, Sieben CJ, Zhong J, et al. Naturally occurring p16(ink4a)-positive cells shorten healthy lifespan. Nature 2016;530:184-9.
DOI: https://doi.org/10.1038/nature16932
Biran A, Zada L, Abou Karam P, Vadai E, Roitman L, Ovadya Y, et al. Quantitative identification of senescent cells in aging and disease. Aging Cell 2017;16:661-71.
DOI: https://doi.org/10.1111/acel.12592
Burd CE, Sorrentino JA, Clark KS, Darr DB, Krishnamurthy J, Deal AM, et al. Monitoring tumorigenesis and senescence in vivo with a p16(ink4a)-luciferase model. Cell 2013;152:340-51.
DOI: https://doi.org/10.1016/j.cell.2012.12.010
Xu P, Wang M, Song WM, Wang Q, Yuan GC, Sudmant PH, et al. The landscape of human tissue and cell type specific expression and co-regulation of senescence genes. Mol Neurodegener 2022;17:5.
DOI: https://doi.org/10.1186/s13024-021-00507-7
Avelar RA, Ortega JG, Tacutu R, Tyler EJ, Bennett D, Binetti P, et al. A multidimensional systems biology analysis of cellular senescence in aging and disease. Genome Biol 2020;21:91.
DOI: https://doi.org/10.1186/s13059-020-01990-9
Xu M, Pirtskhalava T, Farr JN, Weigand BM, Palmer AK, Weivoda MM, et al. Senolytics improve physical function and increase lifespan in old age. Nat Med 2018;24:1246-56.
DOI: https://doi.org/10.1038/s41591-018-0092-9
de Keizer PL. The fountain of youth by targeting senescent cells? Trends Mol Med 2017;23:6-17.
DOI: https://doi.org/10.1016/j.molmed.2016.11.006
Ovadya Y, Krizhanovsky V. Strategies targeting cellular senescence. J Clin Invest 2018;128:1247-54.
DOI: https://doi.org/10.1172/JCI95149
Chaib S, Tchkonia T, Kirkland JL. Cellular senescence and senolytics: The path to the clinic. Nat Med 2022;28:1556-68.
DOI: https://doi.org/10.1038/s41591-022-01923-y
Zhang L, Pitcher LE, Prahalad V, Niedernhofer LJ, Robbins PD. Targeting cellular senescence with senotherapeutics: Senolytics and senomorphics. FEBS J 2023;290:1362-83.
DOI: https://doi.org/10.1111/febs.16350
Demaria M, Ohtani N, Youssef SA, Rodier F, Toussaint W, Mitchell JR, et al. An essential role for senescent cells in optimal wound healing through secretion of pdgf-aa. Dev Cell 2014;31:722-33.
DOI: https://doi.org/10.1016/j.devcel.2014.11.012
Gal H, Lysenko M, Stroganov S, Vadai E, Youssef SA, Tzadikevitch-Geffen K, et al. Molecular pathways of senescence regulate placental structure and function. EMBO J 2019;38:e100849.
DOI: https://doi.org/10.15252/embj.2018100849
Gibaja A, Aburto MR, Pulido S, Collado M, Hurle JM, Varela-Nieto I, et al. Tgfbeta2-induced senescence during early inner ear development. Sci Rep 2019;9:5912.
DOI: https://doi.org/10.1038/s41598-019-42040-0
Ritschka B, Storer M, Mas A, Heinzmann F, Ortells MC, Morton JP, et al. The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration. Genes Dev 2017;31:172-83.
DOI: https://doi.org/10.1101/gad.290635.116
Da Silva-Alvarez S, Guerra-Varela J, Sobrido-Camean D, Quelle A, Barreiro-Iglesias A, Sanchez L, et al. Developmentally-programmed cellular senescence is conserved and widespread in zebrafish. Aging (Albany NY) 2020;12:17895-901.
DOI: https://doi.org/10.18632/aging.103968
Grosse L, Wagner N, Emelyanov A, Molina C, Lacas-Gervais S, Wagner KD, et al. Defined p16(high) senescent cell types are indispensable for mouse healthspan. Cell Metab 2020;32:87-99.e6.
DOI: https://doi.org/10.1016/j.cmet.2020.05.002
Blagosklonny MV. Cell senescence, rapamycin and hyperfunction theory of aging. Cell Cycle 2022;21:1456-67.
DOI: https://doi.org/10.1080/15384101.2022.2054636
Coppe JP, Desprez PY, Krtolica A, Campisi J. The senescence-associated secretory phenotype: The dark side of tumor suppression. Annu Rev Pathol 2010;5:99-118.
DOI: https://doi.org/10.1146/annurev-pathol-121808-102144
da Silva PFL, Ogrodnik M, Kucheryavenko O, Glibert J, Miwa S, Cameron K, et al. The bystander effect contributes to the accumulation of senescent cells in vivo. Aging Cell 2019;18:e12848.
DOI: https://doi.org/10.1111/acel.12848
Nelson G, Wordsworth J, Wang C, Jurk D, Lawless C, Martin-Ruiz C, et al. A senescent cell bystander effect: Senescence-induced senescence. Aging Cell 2012;11:345-9.
DOI: https://doi.org/10.1111/j.1474-9726.2012.00795.x
Cristofalo VJ. Sa beta gal staining: Biomarker or delusion. Exp Gerontol 2005;40:836-8.
DOI: https://doi.org/10.1016/j.exger.2005.08.005
Sikora E, Bielak-Zmijewska A, Mosieniak G. A common signature of cellular senescence; does it exist? Ageing Res Rev 2021;71:101458.
DOI: https://doi.org/10.1016/j.arr.2021.101458
Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 1995;92:9363-7.
DOI: https://doi.org/10.1073/pnas.92.20.9363
Itahana K, Campisi J, Dimri GP. Methods to detect biomarkers of cellular senescence: The senescence-associated beta-galactosidase assay. Methods Mol Biol 2007;371:21-31.
DOI: https://doi.org/10.1007/978-1-59745-361-5_3
Marzullo M, Mai ME, Ferreira MG. Whole-mount senescence-associated beta-galactosidase (sa-beta-gal) activity detection protocol for adult zebrafish. Bio Protoc 2022;12.
DOI: https://doi.org/10.21769/BioProtoc.4457
Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, et al. Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell 2006;5:187-95.
DOI: https://doi.org/10.1111/j.1474-9726.2006.00199.x
Kurz DJ, Decary S, Hong Y, Erusalimsky JD. Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J Cell Sci 2000;113:3613-22.
DOI: https://doi.org/10.1242/jcs.113.20.3613
Van Houcke J, De Groef L, Dekeyster E, Moons L. The zebrafish as a gerontology model in nervous system aging, disease, and repair. Ageing Res Rev 2015;24:358-68.
DOI: https://doi.org/10.1016/j.arr.2015.10.004
Gerhard GS. Comparative aspects of zebrafish (Danio rerio) as a model for aging research. Exp Gerontol 2003;38:1333-41.
DOI: https://doi.org/10.1016/j.exger.2003.10.022
Valenzano DR, Terzibasi E, Cattaneo A, Domenici L, Cellerino A. Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish nothobranchius furzeri. Aging cell 2006;5:275-8.
DOI: https://doi.org/10.1111/j.1474-9726.2006.00212.x
Kishi S, Uchiyama J, Baughman AM, Goto T, Lin MC, Tsai SB. The zebrafish as a vertebrate model of functional aging and very gradual senescence. Exp Gerontol 2003;38:777-86.
DOI: https://doi.org/10.1016/S0531-5565(03)00108-6
Tsai SB, Tucci V, Uchiyama J, Fabian NJ, Lin MC, Bayliss PE, et al. Differential effects of genotoxic stress on both concurrent body growth and gradual senescence in the adult zebrafish. Aging Cell 2007;6:209-24.
DOI: https://doi.org/10.1111/j.1474-9726.2007.00278.x
Novoa B, Pereiro P, Lopez-Munoz A, Varela M, Forn-Cuni G, Anchelin M, et al. Rag1 immunodeficiency-induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress. Aging Cell 2019;18:e13020.
DOI: https://doi.org/10.1111/acel.13020
El Mai M, Marzullo M, de Castro IP, Ferreira MG. Opposing p53 and mtor/akt promote an in vivo switch from apoptosis to senescence upon telomere shortening in zebrafish. Elife 2020;9:e54935.
DOI: https://doi.org/10.7554/eLife.54935
Arslan-Ergul A, Erbaba B, Karoglu ET, Halim DO, Adams MM. Short-term dietary restriction in old zebrafish changes cell senescence mechanisms. Neuroscience 2016;334:64-75.
DOI: https://doi.org/10.1016/j.neuroscience.2016.07.033
Henriques CM, Carneiro MC, Tenente IM, Jacinto A, Ferreira MG. Telomerase is required for zebrafish lifespan. PLoS Genet 2013;9:e1003214.
DOI: https://doi.org/10.1371/journal.pgen.1003214
Van Houcke J, Marien V, Zandecki C, Vanhunsel S, Moons L, Ayana R, et al. Aging impairs the essential contributions of non-glial progenitors to neurorepair in the dorsal telencephalon of the killifish Nothobranchius furzeri. Aging Cell 2021;20:e13464.
DOI: https://doi.org/10.1111/acel.13464
de Bakker DEM, Valenzano DR. Turquoise killifish: A natural model of age-dependent brain degeneration. Ageing Res Rev 2023;90:102019.
DOI: https://doi.org/10.1016/j.arr.2023.102019
Genade T, Benedetti M, Terzibasi E, Roncaglia P, Valenzano DR, Cattaneo A, et al. Annual fishes of the genus nothobranchius as a model system for aging research. Aging Cell 2005;4:223-33.
DOI: https://doi.org/10.1111/j.1474-9726.2005.00165.x
Graf M, Hartmann N, Reichwald K, Englert C. Absence of replicative senescence in cultured cells from the short-lived killifish nothobranchius furzeri. Exp Gerontol 2013;48:17-28.
DOI: https://doi.org/10.1016/j.exger.2012.02.012
Song L, Li C, Wu F, Zhang S. Dietary intake of diosgenin delays aging of male fish Nothobranchius guentheri through modulation of multiple pathways that play prominent roles in ros production. Biogerontology 2022;23:201-13.
DOI: https://doi.org/10.1007/s10522-022-09955-0
Li S, Hou Y, Liu K, Zhu H, Qiao M, Sun X, et al. Metformin protects against inflammation, oxidative stress to delay poly i:C-induced aging-like phenomena in the gut of an annual fish. J Gerontol A Biol Sci Med Sci 2022;77:276-82.
DOI: https://doi.org/10.1093/gerona/glab298
Zhu H, Li X, Qiao M, Sun X, Li G. Resveratrol alleviates inflammation and er stress through sirt1/nrf2 to delay ovarian aging in a short-lived fish. J Gerontol A Biol Sci Med Sci 2023;78:596-602.
DOI: https://doi.org/10.1093/gerona/glad009
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Dev Dyn 1995;203:253-10.
DOI: https://doi.org/10.1002/aja.1002030302
Zupkovitz G, Kabiljo J, Kothmayer M, Schlick K, Schofer C, Lagger S, et al. Analysis of methylation dynamics reveals a tissue-specific, age-dependent decline in 5-methylcytosine within the genome of the vertebrate aging model Nothobranchius furzeri. Front Mol Biosci 2021;8:627143.
DOI: https://doi.org/10.3389/fmolb.2021.627143
Gilbert MJ, Zerulla TC, Tierney KB. Zebrafish (Danio rerio) as a model for the study of aging and exercise: Physical ability and trainability decrease with age. Exp Gerontol 2014;50:106-13.
DOI: https://doi.org/10.1016/j.exger.2013.11.013
McCampbell KK, Springer KN, Wingert RA. Analysis of nephron composition and function in the adult zebrafish kidney. J Vis Exp 2014:e51644.
DOI: https://doi.org/10.3791/51644-v
Gonzalez-Gualda E, Baker AG, Fruk L, Munoz-Espin D. A guide to assessing cellular senescence in vitro and in vivo. FEBS J 2021;288:56-80.
DOI: https://doi.org/10.1111/febs.15570
Gioglio L, Cusella de AM, Boratto R, Poggi P. An improved method for beta-galactosidase activity detection on muscle tissue. A light and electron microscopic study. Ann Anat 2002;184:153-7.
DOI: https://doi.org/10.1016/S0940-9602(02)80009-7
Menke AL, Spitsbergen JM, Wolterbeek AP, Woutersen RA. Normal anatomy and histology of the adult zebrafish. Toxicol Pathol 2011;39:759-75.
DOI: https://doi.org/10.1177/0192623311409597
Dyková I, Zák J, Blazek R, Reichard M, Soucková K, Slaby O. Histology of major organ systems of fishes: Short-lived model species. J Vertebr Biol 2022;71:21074.
DOI: https://doi.org/10.25225/jvb.21074
Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol 1998;11:155-68.
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: An open-source platform for biological-image analysis. Nature Methods 2012;9:676-82.
DOI: https://doi.org/10.1038/nmeth.2019
Borgonovo J, Allende-Castro C, Medinas DB, Cardenas D, Cuevas MP, Hetz C, et al. Immunohistochemical characterisation of the adult nothobranchius furzeri intestine. Cell Tissue Res 2024;395:21-38.
DOI: https://doi.org/10.1007/s00441-023-03845-8
Fathi E, Farahzadi R, Sheikhzadeh N. Immunophenotypic characterization, multi-lineage differentiation and aging of zebrafish heart and liver tissue-derived mesenchymal stem cells as a novel approach in stem cell-based therapy. Tissue Cell 2019;57:15-21.
DOI: https://doi.org/10.1016/j.tice.2019.01.006
Bowley G, Kugler E, Wilkinson R, Lawrie A, van Eeden F, Chico TJA, et al. Zebrafish as a tractable model of human cardiovascular disease. Br J Pharmacol 2022;179:900-17.
DOI: https://doi.org/10.1111/bph.15473
Jensen B, Agger P, de Boer BA, Oostra RJ, Pedersen M, van der Wal AC, et al. The hypertrabeculated (noncompacted) left ventricle is different from the ventricle of embryos and ectothermic vertebrates. Biochim Biophys Acta 2016;1863:1696-706.
DOI: https://doi.org/10.1016/j.bbamcr.2015.10.018
Kenney JW, Steadman PE, Young O, Shi MT, Polanco M, Dubaishi S, et al. A 3D adult zebrafish brain atlas (AZBA) for the digital age. Elife 2021;10:e69988.
DOI: https://doi.org/10.7554/eLife.69988
Huang W, Hickson LJ, Eirin A, Kirkland JL, Lerman LO. Cellular senescence: The good, the bad and the unknown. Nat Rev Nephrol 2022;18:611-27.
DOI: https://doi.org/10.1038/s41581-022-00601-z
Docherty MH, O'Sullivan ED, Bonventre JV, Ferenbach DA. Cellular senescence in the kidney. J Am Soc Nephrol 2019;30:726-36.
DOI: https://doi.org/10.1681/ASN.2018121251
Mohamedien D, Mokhtar DM, Abdellah N, Awad M, Albano M, Sayed RKA. Ovary of zebrafish during spawning season: Ultrastructure and immunohistochemical profiles of sox9 and myostatin. Animals (Basel) 2023;13:3362.
DOI: https://doi.org/10.3390/ani13213362
Xu A, Teefy BB, Lu RJ, Nozownik S, Tyers AM, Valenzano DR, et al. Transcriptomes of aging brain, heart, muscle, and spleen from female and male african turquoise killifish. Sci Data 2023;10:695.
DOI: https://doi.org/10.1038/s41597-023-02609-x
Wang C, Jurk D, Maddick M, Nelson G, Martin-Ruiz C, von Zglinicki T. DNA damage response and cellular senescence in tissues of aging mice. Aging Cell 2009;8:311-23.
DOI: https://doi.org/10.1111/j.1474-9726.2009.00481.x
Tuttle CSL, Waaijer MEC, Slee-Valentijn MS, Stijnen T, Westendorp R, Maier AB. Cellular senescence and chronological age in various human tissues: A systematic review and meta-analysis. Aging Cell 2020;19:e13083.
DOI: https://doi.org/10.1111/acel.13083
Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, et al. Ink4a/arf expression is a biomarker of aging. J Clin Invest 2004;114:1299-307.
DOI: https://doi.org/10.1172/JCI200422475
Cui R, Medeiros T, Willemsen D, Iasi LNM, Collier GE, Graef M, et al. Relaxed selection limits lifespan by increasing mutation load. Cell 2019;178:385-99.e20.
DOI: https://doi.org/10.1016/j.cell.2019.06.004
Anderson R, Lagnado A, Maggiorani D, Walaszczyk A, Dookun E, Chapman J, et al. Length-independent telomere damage drives post-mitotic cardiomyocyte senescence. EMBO J 2019;38:e100492.
DOI: https://doi.org/10.15252/embj.2018100492
Mehdizadeh M, Aguilar M, Thorin E, Ferbeyre G, Nattel S. The role of cellular senescence in cardiac disease: Basic biology and clinical relevance. Nat Rev Cardiol 2022;19:250-64.
DOI: https://doi.org/10.1038/s41569-021-00624-2
Chen MS, Lee RT, Garbern JC. Senescence mechanisms and targets in the heart. Cardiovasc Res 2022;118:1173-87.
DOI: https://doi.org/10.1093/cvr/cvab161
Ethics Approval
The animal experiment was approved by the Austrian Federal Ministry of Education, Science and ResearchSupporting Agencies
Austrian Science Fund (FWF)/Herzfelder’sche FamilienstiftungHow to Cite
Schöfer, S., Laffer, S., Kirchberger, S., Kothmayer, M., Löhnert, R., Ebner, E. E., Weipoltshammer, K., Distel, M., Pusch, O., & Schöfer, C. (2024). Senescence-associated ß-galactosidase staining over the lifespan differs in a short- and a long-lived fish species. European Journal of Histochemistry, 68(1). https://doi.org/10.4081/ejh.2024.3977
Copyright (c) 2024 The Author(s)
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
PAGEPress has chosen to apply the Creative Commons Attribution NonCommercial 4.0 International License (CC BY-NC 4.0) to all manuscripts to be published.
