The Identification of Zebrafish Mutants Showing Alterations in Senescence-Associated Biomarkers

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms.

[1]  I. Zhdanova,et al.  Aging of the circadian system in zebrafish and the effects of melatonin on sleep and cognitive performance , 2008, Brain Research Bulletin.

[2]  Tilman Grune,et al.  Lipofuscin: formation, distribution, and metabolic consequences. , 2007, Annals of the New York Academy of Sciences.

[3]  E. Rodriguez-Boulan,et al.  The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells , 2007, Proceedings of the National Academy of Sciences.

[4]  M. Mattson,et al.  Telomere Protection Mechanisms Change during Neurogenesis and Neuronal Maturation: Newly Generated Neurons Are Hypersensitive to Telomere and DNA Damage , 2007, The Journal of Neuroscience.

[5]  D. Neuberg,et al.  Differential effects of genotoxic stress on both concurrent body growth and gradual senescence in the adult zebrafish , 2007, Aging cell.

[6]  Valter Tucci,et al.  Cognitive Aging in Zebrafish , 2006, PloS one.

[7]  M. Beal,et al.  Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases , 2006, Nature.

[8]  Nancy Hopkins,et al.  Mutagenesis strategies in zebrafish for identifying genes involved in development and disease. , 2006, Trends in genetics : TIG.

[9]  Antonino Cattaneo,et al.  Temperature affects longevity and age‐related locomotor and cognitive decay in the short‐lived fish Nothobranchius furzeri , 2006, Aging cell.

[10]  D. DiMaio,et al.  Senescence‐associated β‐galactosidase is lysosomal β‐galactosidase , 2006 .

[11]  M. Mattson,et al.  TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons , 2006, Journal of neurochemistry.

[12]  S. Kishi.  Zebrafish as Aging Models , 2006 .

[13]  D. DiMaio,et al.  Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. , 2006, Aging cell.

[14]  G. Stoica,et al.  ATM deficiency induces oxidative stress and endoplasmic reticulum stress in astrocytes , 2005, Laboratory Investigation.

[15]  H. Maaswinkel,et al.  Behavioral screening for nightblindness mutants in zebrafish reveals three new loci that cause dominant photoreceptor cell degeneration , 2005, Mechanisms of Ageing and Development.

[16]  Paola Roncaglia,et al.  Annual fishes of the genus Nothobranchius as a model system for aging research , 2005, Aging cell.

[17]  S. Lowe,et al.  p63 deficiency activates a program of cellular senescence and leads to accelerated aging. , 2005, Genes & development.

[18]  Douglas L. Schmucker,et al.  Age-related changes in liver structure and function: Implications for disease ? , 2005, Experimental Gerontology.

[19]  T. Lange,et al.  DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion , 2005, Nature Cell Biology.

[20]  M. Boulton,et al.  RPE lipofuscin and its role in retinal pathobiology. , 2005, Experimental eye research.

[21]  J. Woulfe,et al.  Lipofuscin and aging: a matter of toxic waste. , 2005, Science of aging knowledge environment : SAGE KE.

[22]  T. de Lange,et al.  Shelterin: the protein complex that shapes and safeguards human telomeres. , 2005, Genes & development.

[23]  Nancy Hopkins,et al.  Identification of 315 genes essential for early zebrafish development. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Evan T Keller,et al.  The use of mature zebrafish (Danio rerio) as a model for human aging and disease. , 2004, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[25]  J. Andersen,et al.  Oxidative stress in neurodegeneration: cause or consequence? , 2004, Nature Reviews Neuroscience.

[26]  S. Kishi.  Functional Aging and Gradual Senescence in Zebrafish , 2004, Annals of the New York Academy of Sciences.

[27]  Colin L. Masters,et al.  Neurodegenerative diseases and oxidative stress , 2004, Nature Reviews Drug Discovery.

[28]  S. Boncompagni,et al.  The contribution of reactive oxygen species to sarcopenia and muscle ageing , 2004, Experimental Gerontology.

[29]  H. Maaswinkel,et al.  ENU-induced late-onset night blindness associated with rod photoreceptor cell degeneration in zebrafish , 2003, Mechanisms of Ageing and Development.

[30]  R. Bryson-Richardson,et al.  Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo , 2003, Development.

[31]  G. Gerhard.  Comparative aspects of zebrafish (Danio rerio) as a model for aging research , 2003, Experimental Gerontology.

[32]  Junzo Uchiyama,et al.  The zebrafish as a vertebrate model of functional aging and very gradual senescence , 2003, Experimental Gerontology.

[33]  D. Watters.  Oxidative stress in ataxia telangiectasia , 2003, Redox report : communications in free radical research.

[34]  C. Deng,et al.  Senescence, aging, and malignant transformation mediated by p53 in mice lacking the Brca1 full-length isoform. , 2003, Genes & development.

[35]  G. Davis,et al.  Unrestricted Synaptic Growth in spinster—a Late Endosomal Protein Implicated in TGF-β-Mediated Synaptic Growth Regulation , 2002, Neuron.

[36]  U. Brunk,et al.  Lipofuscin: mechanisms of age-related accumulation and influence on cell function. , 2002, Free radical biology & medicine.

[37]  C. Kasales,et al.  Life spans and senescent phenotypes in two strains of Zebrafish (Danio rerio) , 2002, Experimental Gerontology.

[38]  E. Porta.  Pigments in Aging: An Overview , 2002, Annals of the New York Academy of Sciences.

[39]  Eli Carmeli,et al.  The biochemistry of aging muscle , 2002, Experimental Gerontology.

[40]  U. Brunk,et al.  The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis. , 2002, European journal of biochemistry.

[41]  D. Yamamoto,et al.  Zebrafish yolk‐specific not really started (nrs) gene is a vertebrate homolog of the Drosophila spinster gene and is essential for embryogenesis , 2002, Developmental dynamics : an official publication of the American Association of Anatomists.

[42]  T. Lange.  Protection of mammalian telomeres , 2002, Oncogene.

[43]  M. Fishman,et al.  From Zebrafish to human: modular medical models. , 2002, Annual review of genomics and human genetics.

[44]  L. Zon,et al.  The art and design of genetic screens: zebrafish , 2001, Nature Reviews Genetics.

[45]  R. Ueda,et al.  Mutations in the Novel Membrane Protein Spinster Interfere with Programmed Cell Death and Cause Neural Degeneration inDrosophila melanogaster , 2001, Molecular and Cellular Biology.

[46]  Masafumi Nakamura,et al.  Telomeric protein Pin2/TRF1 induces mitotic entry and apoptosis in cells with short telomeres and is down-regulated in human breast tumors , 2001, Oncogene.

[47]  G. Gerhard,et al.  Molecular cloning and sequence analysis of the Danio rerio catalase gene. , 2000, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[48]  N. Holbrook,et al.  Oxidants, oxidative stress and the biology of ageing , 2000, Nature.

[49]  D. Kurz,et al.  Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. , 2000, Journal of cell science.

[50]  L. Schmued,et al.  Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration , 2000, Brain Research.

[51]  J. Shaw,et al.  Cloning and expression of a cDNA coding for catalase from zebrafish (Danio rerio). , 2000, Journal of agricultural and food chemistry.

[52]  J. Dowling,et al.  Disruption of the Olfactoretinal Centrifugal Pathway May Relate to the Visual System Defect in night blindness bMutant Zebrafish , 2000, The Journal of Neuroscience.

[53]  A. Amsterdam,et al.  A large-scale insertional mutagenesis screen in zebrafish. , 1999, Genes & development.

[54]  J. Morrow,et al.  Loss of the ataxia-telangiectasia gene product causes oxidative damage in target organs. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[55]  J. Griffith,et al.  Mammalian Telomeres End in a Large Duplex Loop , 1999, Cell.

[56]  Bas van Steensel,et al.  TRF2 Protects Human Telomeres from End-to-End Fusions , 1998, Cell.

[57]  U. Brunk,et al.  Lipofuscin: Mechanisms of formation and increase with age , 1998, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[58]  J. Dowling,et al.  A dominant form of inherited retinal degeneration caused by a non-photoreceptor cell-specific mutation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[59]  Y. Shiloh,et al.  Ataxia-telangiectasia: is ATM a sensor of oxidative damage and stress? , 1997, BioEssays : news and reviews in molecular, cellular and developmental biology.

[60]  R. Weindruch,et al.  Oxidative Stress, Caloric Restriction, and Aging , 1996, Science.

[61]  C Roskelley,et al.  A biomarker that identifies senescent human cells in culture and in aging skin in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[62]  C. Kimmel,et al.  Stages of embryonic development of the zebrafish , 1995, Developmental dynamics : an official publication of the American Association of Anatomists.

[63]  R. Jolly,et al.  Lipofuscin in bovine muscle and brain: a model for studying age pigment. , 1995, Gerontology.

[64]  C. Nüsslein-Volhard,et al.  Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate , 1994, Current Biology.

[65]  R. S. Sohal,et al.  Comparison of mitochondrial pro-oxidant generation and anti-oxidant defenses between rat and pigeon: possible basis of variation in longevity and metabolic potential , 1993, Mechanisms of Ageing and Development.

[66]  K. Kikugawa,et al.  Fluorescent and cross-linked proteins formed by free radical and aldehyde species generated during lipid oxidation. , 1989, Advances in experimental medicine and biology.