The bony fishes (Osteichthyes) represent the largest class of vertebrates, with some 24,000 extant species. However, only limited data are available on aging and senescence of just a few of these species (Patnaik et al., 1994). Several small tropical species, such as the guppy (Lebistes reticularis) (Reznick et al., 2001; Reznick, 1997), and a species of annual fish (Cynelobias bellottii) (Valdesalici and Cellerino, 2003), manifest increased mortality with age typical of gradual senescence and definite lifespan (Belinsky et al., 1997; Finch and Austad, 2001; Finch and Ruvkun, 2001). In addition, age-related degenerative changes characteristic of gradual senescence, such as loss of muscle fibers, endocrine abnormalities, decline in reproductive capacity, increased cancer incidence, and increases in various pathological lesions, have been documented in other fish species (Patnaik et al., 1994). In comparison to these fish, the aging process of zebrafish has not yet been addressed adequately.
A number of aging theories have been proposed, including oxidative or genotoxic stress and damage, telomere metabolism, regulation of caloric restriction (CR), and control of metabolic energy rate. A growing body of evidence suggests that reactive oxygen species (ROS) may regulate the aging process and may participate in cellular replicative senescence. Consistent with a role for ROS in senescence, examination in cell-culture systems showed that older cells have higher levels of ROS than younger ones (Finkel and Holbrook, 2000). Moreover, treatment with sublethal concentrations of hydrogen peroxide induces a senescence-like phenotype in primary human and rodent fibroblast (Chen and Ames, 1994; Sohal and Weindruch, 1996; Wolf et al., 2002), termed ''stress-induced premature senescence'' (Toussaint et al., 2000). On the other hand, antioxidant treatment or low-oxygen condition prolongs the lifespan of cells (Finkel and Holbrook, 2000). The free radical theory of aging, which proposes that oxidative stress increases with age, may provide some clues to this phenomenon. Specifically, reactive oxygen intermediates (ROI) act as second messengers culminating in activation of signal transduction pathways and subsequently changes in the activity of various transcription factors. Another theory holds that cellular replicative senescence is regulated by factors that govern the shortening of telo-meres in dividing cells of the adult tissues and organs.
Telomeric TTAGGG repeats in vertebrates, which protect chromosome ends, are shortened with each division of most cells in the adult due to lack of telomerase activity (Blackburn, 2001). However, compelling evidence indicates that the ability of telomerase to elongate telomeres is regulated by several other telomere-binding factors with multiple interacting proteins, which construct a huge "telosome" complex (Blackburn, 2001; Liu et al., 2004; Rodier et al., 2005).
The CR paradigm provides additional support for this hypothesis. CR, which decreases oxidative stress, delays the appearance of expression of several age-associated genes (Sohal and Weindruch, 1996). Use of high-density oligonucleotide arrays and genome-wide transcript profiles in mice and flies revealed that aging was accompanied by a differential gene expression pattern with indications of a marked stress response and lower expression of metabolic and biosynthetic genes (Lee et al., 1999; Pletcher et al., 2002; Zou et al., 2000). Most alterations were either completely or partially prevented by caloric restriction, the only intervention known to retard aging in mammals. Transcriptional patterns of calorie-restricted animals suggest that caloric restriction retards the aging process by causing a metabolic shift toward increased protein turnover and decreased macro-molecular damage. On the other hand, oxidative damage is repaired less well in telomeric DNA than elsewhere in the chromosome, and oxidative stress accelerates telomere loss, whereas antioxidants decelerate it. Therefore, oxida-tive stress is also an important modulator of telomere loss, and telomere-driven replicative senescence is primarily a stress response. Among oxidative stress, regulation of CR, and telomere metabolism, there also seems to be a significant cross-talk mechanism with each other in the course of the aging process. However, genetic evidence of this mechanism in higher vertebrates has not been elucidated yet.
The propagation of a species could depend in principle either on the unlimited maintenance of its individuals or on the ability to renew the population with young members. Nature most commonly has chosen the latter option. Renewal is typically achieved by the setting aside of a pristine lineage of genetic information in the germ line, which is passed on by sexual reproduction. In contrast, the somatic lineage of all animals declines and degenerates with age, giving rise to phenotypic changes recognized as aging. Studies in model organisms have begun to map out important genes and pathways that seem to regulate the pace of aging and that are remarkably conserved from yeast to worm and insect. These studies have the great advantage that one can use genetic approaches to search directly for mutations that change lifespan. This makes it possible to identify mechanisms in a way that is independent of any preconceived model of aging. Recently, the analysis of such mutations that affect lifespan has revealed several different pathways that influence the aging process. These include, notably, signaling molecules from the Insulin-like growth factor-1 (IGF-1)-FoxOs (the FoxO subfamily of forkhead transcription factors) pathway which are highly conserved in evolution and have been identified in yeast, flies, and worms as regulating aging (Guarente and Picard, 2005; Kenyon, 2005; Murphy et al., 2003). Thus once a gene has been identified as regulating aging in lower organisms it can be tested in mice by knockout or transgenic technologies. However, our inability to do forward genetics in vertebrates places serious restrictions on the types of genes we might find. Moreover, it seems quite likely that genetic screening in invertebrates will miss genes affecting cellular proliferation in adults, which confers on mammals with their relatively longer lifespan. Additionally, those unique genes and their functions related to longevity in vertebrates are not likely to be found in unicellular organisms and other invertebrate model systems. To address these questions in a more tractable and high-throughput setting, alternative vertebrate model systems are needed. Since zebrafish offer a number of advantages for the study of diseases and for tissue and organ development, we begun utilizin zebrafish as a new vertebrate model to explore the aging and senescence process with pathophysiological phenotypes. As part of this ongoing effort, mutagenesis and transgenesis approaches are expected to be extremely useful.
Zebrafish have proven to be an outstanding animal model system for studying vertebrate development using both genetic and genomic approaches. For instance, large-scale chemical mutagenesis strategies have been used to identify numerous zebrafish mutants with developmental abnormalities or defects. However, current approaches lack the ability to demonstrate developmental imprinting and identify adult or late-age onset mutants that can perturb the aging process. Although C. elegans and Drosophila are well-established aging models which have been used to identify a number of single gene mutations resulting in extended or shortened lifespan, these relatively shorter-lived invertebrates are unlikely to provide information on essential genes and molecular mechanisms that are unique to longer-lived vertebrates as described above. For instance, it is difficult to address the organo-specific aging process in nervous, cardiovascular, immune, and musculoskeletal system of invertebrates. Moreover, invertebrates cannot be utilized to explore developmental and aging functions of vertebrate-specific features such as the kidney, a multichambered heart, multilineage hematopoiesis, a notochord, and neural crest cells.
Mutagenesis is one of the best approaches for identifying a gene function. Of vertebrates studied to date, mutations can be generated and recovered most readily in zebrafish. Furthermore, as a result of recent advances in technology, zebrafish can be utilized for both forward genetics and, importantly, reverse genetics. For example, transgenesis coupled with chemical mutagenesis can be employed for the analysis of promoter activity and specificity utilizing an effective in vivo reporter system such as GFP (Dodd et al., 2000). It is also possible to use high-throughput transgenic approaches such as GFP-positive embryos to study the function of gene products by taking advantage of the transparency of fish during its developmental stages. More recently, the Targeting Induced Local Lesions in Genomes (TILLING) approach coupled with a direct resequencing strategy of a target gene in mutagenized genomic DNA has been developed in the zebrafish system (Henikoff et al., 2004; Wienholds et al., 2002; Wienholds et al., 2003). This approach, along with other related methods, brings zebrafish reverse genetics to a level equivalent with that of mice. Therefore, functional genetic and genomic analysis of signaling pathways involved in aging of zebrafish will be readily possible, once we identify effective and robust aging markers.
The zebrafish system is already well established in developmental biology, and recently zebrafish models have been used in disease-based biomedical studies. In marked contrast, the aging process of zebrafish has not been significantly addressed, so that there is little known with respect to the aging process and cellular senescence in zebrafish. Susceptibility to most human chronic diseases is affected by the aging process. Therefore, it is crucial to know how zebrafish aging is compared with aging in mammals, particularly since we also view zebrafish as a useful surrogate model for multiple human diseases. Moreover, through comparative studies of aging among vertebrates, we may be able to learn how we can take advantage of the certain unique properties in lower vertebrates, such as multiple organs' regeneration and indeterminate growth, which have been lost during mammalian evolution.
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