Using Zebrafish Models to Explore Genetic and Epigenetic Impacts On

Using zebraﬁsh models to explore genetic and epigenetic impacts on evolutionary developmental origins of aging

SHUJI KISHI

JUPITER, FLA

Can we reset, reprogram, rejuvenate, or reverse the organismal aging process? Certain genetic manipulations could at least reset and reprogram epigenetic dynamics beyond phenotypic plasticity and elasticity in cells, which can be manipu- lated further into organisms. However, in a whole complex aging organism, how can we rejuvenate intrinsic resources and infrastructures in an intact and noninvasive manner? The incidence of diseases increases exponentially with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging- associated diseases are sporadic but essentially inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these 2 phenomena to rejuvenate over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states based on diverse epigenotypes in response to intrinsic or extrinsic environmental cues and genetic perturbations. We hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds and windows of plasticity and its robustness by molecular genetic and chemical epigenetic approaches, we have successfully conducted experiments to isolate zebraﬁsh mutants expressing apparently altered senescence phenotypes during their embryonic and/or larval stages (‘‘embryonic/larval senescence’’). Subsequently, at least some of these mutant animals were found to show a shortened life span, whereas others would be expected to live longer into adulthood. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other hand, unexpected senescence-related genes might also be involved in the early developmental process and its regulation. The ease of manipulation using the zebraﬁsh system allows us to conduct an exhaustive exploration of novel genes, genotypes, and epigenotypes that can be linked to the senescence phenotype, which facilitates searching for the evolutionary and developmental origins of aging in vertebrates. (Translational Research 2014;163:123–135)

Abbreviations: SA-b-gal ¼ Senescence-associated b-galactosidase; TERT ¼ Telomerase reverse transcriptase

From the Department of Metabolism & Aging, The Scripps Research Submitted for publication August 22, 2013; revision submitted Institute, Scripps Florida, Jupiter, Fla. October 20, 2013; accepted for publication October 21, 2013. Conﬂicts of Interest: The author has read the journal’s policy on con- Reprint requests: Shuji Kishi, MD, PhD, Department of Metabolism & ﬂicts of interest and have none to declare. Aging, The Scripps Research Institute, Scripps Florida, 130 Scripps The work of the author’s laboratory reviewed in this article was funded Way, #3B3, Jupiter, FL 33458; e-mail: [email protected]. by research grants from The Ellison Medical Foundation, the Glenn 1931-5244/$ - see front matter Foundation for Medical Research, the A-T Children’s Project, and Ó 2014 Mosby, Inc. All rights reserved. the National Institute of General Medical Sciences/National Institute http://dx.doi.org/10.1016/j.trsl.2013.10.004 on Aging/National Institutes of Health.

123 Translational Research 124 Kishi February 2014

The mechanisms of aging are currently the focus of life span has not proved to be universally true, as shown extensive investigations throughout the world. During in several cases including humans, some fish species, the aging process, multiple forms of tissue- and organ- and honeybees (Apis mellifera).3-9 Therefore, a cross- specific damage and pathophysiological change accu- species comparative biology of aging prospective is mulate and, accordingly, a number of chronic diseases necessary to understand and obtain a more complete appear with advancing age. Scientists are now looking picture for fundamental causes of aging in speciation for the principles governing the ubiquitous process of and biodiversity. aging to find, ultimately, novel ways to attenuate or Development is obviously under the precise control delay aging in humans as well as to develop interven- of genetic mechanisms programmed in the genome hav- tions for age-associated diseases. However, understanding plasticity, yet through remodeling of epigenomes ing the molecular mechanisms of aging in vertebrates is (Fig 1). However, such precise genetic programming, still a major challenge of modern biology and biomed- unfolded by epigenetic interventions, may still be ical science. There are various animal models to be disturbed later during the aging process and may un- considered and used in aging studies, and determining dergo many stochastic deteriorative challenges against the optimal model system or systems remains one of regenerative/repair responses and restorations when the most important issues. Given certain limitations in the reactivation of certain phases of the early develop- each and all the existing models of human aging, calling mental program/process contributes to and/or are for an integrative approach that uses diverse species in required (Fig 1). When we consider aging as an exten- the hope that each can provide a piece of the puzzle sion of the biologic process of development, the power and, together, would help to identify critical elements of developmental biology and genetics in zebrafish be- common to aging in all organisms, would be ideal. comes a monumental and instructive wealth of informa- One of the essential limitations is availability of ge- tion and exploration. Zebrafish can be used extensively netic/genomic and epigenetic/epigenomic information in searching for evolutionary and developmental origins detailed on relevant species. This provides the rationale of aging common in vertebrates. In fact, some of the for current biomedical investigations into the mecha- genes we identified by an apparent senescence pheno- nisms of aging being conducted concurrently in geneti- type during embryogenesis (‘‘embryonic senescence’’) cally robust species like worms, fruit flies, and mice. In had already been associated with cellular senescence fact, small and prolific invertebrates, such as Caeno- and chronological aging in other organisms, whereas rhabditis elegans and Drosophila melanogaster, can many others still wait to be linked to the conventional provide a strong basis for unbiased genetic screens aging process in future studies. Complete loss of func- that identify and determine the precise functions of tion of developmentally essential genes induces embry- novel genes and their epigenetic profiles that regulate onic (or larval) lethality, whereas it seems like their aging and life span transgenerationally and evolution- partial loss of function (ie, decrease of function by het- ally. This approach has already led to the successful erozygote or hypomorphic mutation) still remains suffi- identification of key genes and pathways evolutionarily cient to go through the early developmental process conserved in and associated with the aging process. because of its plasticity or, rather, heterozygote advan- While evolutionally conserved mechanisms of aging tage. In some cases, however, such partial loss of func- could play roles in various organisms, manifestations tion of genes compromises normal homeostasis as a of aging can also differ in different species. For result of a heterozygote disadvantage (‘‘haploinsuffi- instance, vertebrates from fish through humans often ciency’’) later in adult life, presenting a number of envi- die of cancer or stroke, but invertebrates such as worms ronmental or epigenetic challenges that less plasticity and insects unlikely die of either. Thus far, only mice can no longer adjust to compensate (Fig 2). In contrast, have served as a widely used genetic model system to any heterozygote-advantageous genes might gain study the aging process in vertebrates, providing impor- certain benefits (much more fitness) by such partial tant insights into mammalian aging as an indispensable loss of function later in life (Fig 2). animal model.1 However, studying only a single genetic Physiological senescence may arise evolutionarily model may be limiting because of the diversified from both genetic and epigenetic drift as well as from complexity of vertebrate aging across species. Some losing developmental plasticity in face of stress signals mechanisms of aging could be conserved evolutionarily from the external environment that interact with func- and publicly in vertebrates, but there are also more tions of multiple genes rather than effects of only a sin- lineage-specific or private mechanisms of aging from gle gene mutation or defect. We wish to identify a a comparatively biologic point of view.2 Even the com- number of such critical genes promptly in a comprehen- mon notion that caloric (or dietary) restriction extends sive manner by using the zebrafish model system. Translational Research Volume 163, Number 2 Kishi 125

Fig 1. Programmed and stochastic regulations of development and aging through linking a genome to epigenomes. Development is pri- marily under the precise control of genetic mechanisms ‘‘pro- Fig 2. Modeling of the impacts of gene mutations results in altered grammed’’ in genomes having plasticity yet through remodeling of (half) dosages of particular gene functions and leads to altered senes- epigenomes. Such precise genetic programming, unfolded by epige- cence outcomes through the aging process. In the schematic, the netic interventions, may still be disturbed later during the continued slope of the solid black line illustrates the age-dependent regression aging process and may undergo many ‘‘stochastic’’deteriorative chal- of accuracy in gene function in a wild-type (Wt) organism resulting, lenges against regenerative/repair responses and restorations when re- in part, from ‘‘programmed’’ senescence, accumulated structural activation of certain phases of the early developmental program/ cellular damage following intrinsic and extrinsic environmental ‘‘sto- process contributes or is required. The association between early chastic’’ events, desynchronization of molecular mechanisms, and so development and late-onset disease with advancing age is thought to on. The slope of the gray or green line shows the age-dependent come from a consequence of developmental plasticity, the phenome- decline of accuracy in gene function in a heterozygous (Het) mutant non by which 1 genome can give rise to a range of physiologically organism identiﬁed in the screening—in other words, the organisms or morphologically adaptive states by remodeling epigenomes (as with a presumed 50% loss of original gene function at birth—leading shown hypothetically in the reversible remodeling of histone methyl- to a shorter (heterozygote disadvantage) or longer (heterozygote ation [Me] and acetylation [Ac]), in response to intrinsic or extrinsic advantage) life span (on the x-axis). Dotted horizontal lines depict environmental cues and genetic perturbations. The primary problem the level of gene function at which certain aging biomarkers can is a decrease, failure, or loss of the adaptive epigenetic response at be documented (ie, aging biomarker thresholds A and B). Homozy- later organismal ages. gous (Homo) mutant (Mu) organisms are found to be lethal early (see x-axis).

Presumably, certain critical portions of aging phenomena may become readily predictable based on advanced allowing for investigation of the intrinsic mechanisms genetic and genomic approaches in zebrafish, and a sys- underlying normal physiological and pathologic tems biology approach will allow us to simulate the ag- events in this organism. Consequently, of the ing process by modeling potential stochastic values, vertebrates studied to date, zebrafish offer the most including epistatic and epigenetic impacts. cost-effective opportunity for high-throughput screens using robotic systems for evaluating mutants, identifying genes, and testing chemicals in vertebrate organ- ZEBRAFISH AS A VERTEBRATE MODEL OF AGING isms.26-28 Zebrafish have emerged as a highly promising model In terms of the life span of zebrafish, however, they for studies of vertebrate aging.10-18 Combining their live quite long—approximately 3 years on average optical transparency at embryonic and larval stages (in (maximum, more than 5 years) in laboratory condi- some cases, even in adults throughout their life tions—and show gradual senescence that is similar in span19), the small size and high fecundity of zebrafish many aspects to humans.10,12 With age, zebrafish show have made them a favorite vertebrate of developmental diversified phenotypes and symptoms, and humans biologists. This has resulted in detailed characterization make them as well. For instance, zebrafish often of the zebrafish genome, the development of multiple display spinal curvature that is possibly related to mutant and transgenic phenotypes, and the emergence muscle abnormalities.12 We also detected senescence- of various molecular genetics techniques (eg, most associated b-galactosidase (SA-b-gal) activity in skin recently, a genome-editing approach and targeted- in aging zebrafish.10,15,29 Moreover, as zebrafish age, gene knockout using nucleases as well as authentic they show increased accrual of lipofuscin (aging knockdown using morpholino antisense oligos),20-25 pigment) in the liver and accumulation of oxidized Translational Research 126 Kishi February 2014 proteins in muscle,10,29 similar to that reported in mice are responsible transgenerationally for the develop- and humans.30-32 Aged zebrafish develop lipofuscin mental origins of aging and disease in vertebrates. accumulation further and drusen-like lesions in retinal There may be remarkably complex interactions among pigment epithelium, which is similar to that seen in unexpected, yet-unidentified, and/or even apparently age-related macular degeneration in humans. Last, unrelated genes, requiring unbiased comprehensive almost all zebrafish develop cataracts as long as they sur- methods of detection and systems biology approaches vive to 4 years of age (unpubl. obs.), when the majority to their identifications. In most vertebrates, unbiased of the oldest fish show retinal atrophy.29 screens for aging mutants are difficult technically and Our studies have further revealed several age-related therefore have not yet been done, even in mice. On degenerative changes and increases in a variety of path- the other hand, specific phenotype-based and unbiased ologic lesions in aging zebrafish, as well as an forward genetic screens in early development for multi- age-dependent decline in both their reproductive and ple biologic and biomedical purposes have already been regenerative capacity.10,15,29 In adult zebrafish, fin carried out successfully in zebrafish and can be adapted regeneration is a highly orchestrated processes involv- readily to current aging research, once any adequate ing wound healing, establishment of the wound senescence biomarker is identified and can be applied epithelium, recruitment of the blastema from mesen- to this model system, particularly in embryos and/or chymal cells underlying the wound epithelium, and larvae during the developmental process. differentiation and outgrowth of the regenerated The features that distinguish the zebrafish as a high- cells.33 More important, such regenerative ability de- throughput, amenable organism for high-content or clines with age, leading to impaired morphology and large-scale screens include their small body size, trans- distorted fin shape,15 which may become a valuable parent embryogenesis, and high reproductive rate, al- hallmark of aging in this animal model. There have lowing the simultaneous evaluation of many siblings also been observed age-associated alterations in circa- throughout their life span under controlled conditions, dian rhythm, sleep, and cognitive function in zebra- with and without certain environmental and pharmaco- fish.13-15,34 Taken together, although zebrafish have a logic challenges. Moreover, the existence of various ze- relatively long life expectancy that might be tedious in brafish mutants and traceable transgenic lines showing some long-term experiments during the simply chrono- particular phenotypes already, including retroviral logical aging process, our observations and other cumu- insertional mutant collections,37 provides invaluable relative evidence suggest that zebrafish have become a sources for the identification of both physiological and great model system to investigate the mechanisms un- pathologic aging characteristics. derlying gradual senescence in vertebrates, and can also serve as a promising model of human aging.10,29 THE AGING PROCESS MIMICKED BY EMBRYONIC/ LARVAL SENESCENCE PHENOTYPES HIGH-THROUGHPUT SCREENING OF SENESCENCE- Humans develop and age very gradually compared ASSOCIATED MUTANTS IN VERTEBRATES with most of the animals used as biomedical research In addition to studying the function of specific genes model systems. The time-consuming aspect of aging that have already been linked to the aging process, it is research may become a critical limitation of the current critically important to conduct unbiased mutant screens study and approach, because some of the aging pro- for aging phenotypes in vertebrates to search for addi- cesses common to humans might, indeed, require a pro- tional genes that affect late-onset diseases or age- longed life span to become apparent and to elucidate related dysfunctions. The mechanisms connecting the adequately the essential determining factors of the organismal aging process with multiple, chronic age- gradual nature and mechanisms of aging. associated human diseases (geriatric diseases) remain Recent studies indicate that the rate of aging can be a complex enigma, and scientists sometimes face con- modulated by environmental/epigenetic as well as ge- flicting arguments about aging phenotypes vs diseases. netic factors, and that the clock of aging can be for- Both intrinsic gene-gene interactions (epistasis) and warded flexibly or even reversed by restoring gene-environment interactions, influencing genome/ge- characteristics of youthfulness to aged cells, tissues, notype and epigenome/epigenotype through numerous and organisms.38 Although certain genetic mutations environmental and epigenetic factors, affect the aging can presumably lead to the structural and functional process and disease progression.35,36 It is poorly decline of various tissues by impairing cellular func- understood, however, which of the complex genetic tions, it is still tough to explain that single genetic mu- elements affect life span evolutionarily, how they are tations alone can account for the entire characteristic correlated with disease susceptibility, and how changes during the aging process shared across individ- stochastic environmental and epigenetic modulations uals within a species and even across species. Therefore, Translational Research Volume 163, Number 2 Kishi 127 the hypothesis that environmental challenge-associated enhancer mutants of type I genes by haploinsufficiency; epigenetic alterations underlie age-related phenotypic Fig 3). This applies to cases when gene mutations lead changes of cells and tissue, as well as the age- to an augmented response to stress (in type I genes) dependent increase in susceptibility to many diseases or, conversely, a resistance to it (in type II genes). in organisms, has gained considerable support. On the Thus, a mutant phenotype in homozygosity as well as other hand, to extend comprehensive explorations and a specific stress challenge (instead of an actual aging analyses, the lengthy life span of experimental animals challenge) in heterozygosity during early development represents a practical obstacle for undertaking either could mimic the real aging process and help to predict cross-sectional or longitudinal aging research, particu- the genotypes that might be susceptible or resistant to larly in genome- and epigenome-wide studies as well homeostatic deteriorations that occur during aging. By as in high-throughput screens of candidate factors. using a chemical sensitizer (eg, hydrogen peroxide) to One of the most promising approaches to confronting mimic environmental influences and/or epigenetic mod- this dilemma is to assess environmentally mimicked ulation, we could indeed identify mutants with an young animals (even embryos and larvae) for reliable altered response to oxidative stress; that is, we expected and easily measurable biomarkers of senescence that the chemical sensitizer to induce heterozygote advan- ‘‘predict’’ an actual (both chronologic and biologic) ag- tage or disadvantage impacts in many of the potential ing phenotype that appears later in life. The causative target genes. We then denoted this chemical genetic link between early development and late-onset disorder methodology CASH (chemically assisted screening in with advancing age is presumably a result of a conse- heterozygotes).29 quence of developmental plasticity, the phenomenon One obvious biomarker of aging to use in unbiased by which one genome can give rise to a range of phys- screens is SA-b-gal, an indicator of cellular senescence iologically and/or morphologically adaptive states in vitro as well as of organismal aging in vertebrates.40-45 based on divergent epigenomes in response to internal In fact, we detected SA-b-gal activity in the skin as well or external environmental cues. Thus, we hypothesized as oxidized protein accumulation in the muscle of aging that the future aging process can be predictive based on zebrafish,10,15,29 similar to that demonstrated in aging adaptivity during the early developmental period. humans.40 We used this marker in a series of screens Modulating the thresholds and windows of plasticity for embryonic senescence phenotypes using more and its robustness by molecular genetic and chemical than 500 mutant genomes from retrovirus-mediated epigenetic approaches, we conducted zebrafish mutant insertional zebrafish mutant lines and others induced screens by identifying apparently altered senescence by N-ethyl-N-nitrosourea chemical mutagenesis.29,37 phenotypes during their embryonic and/or larval stages Because all the 306 insertional mutations screened (embryonic/larval senescence). were ultimately homozygous lethal, we needed to In genotypes susceptible to accelerated aging (caused explore the effects of missing just one copy of the by a heterozygote disadvantage) or even delayed aging genes in heterozygous aging adult fish. However, (caused by a heterozygote advantage), such biomarkers instead of characterizing the aging phenotypes might manifest in young animals with certain gene mu- throughout their life span, we first examined which tations (or alleles), either spontaneously or after stress of these mutants showed altered SA-b-gal activity (Fig 3). Even if such mutations are lethal in homozy- during embryonic development within 5 days gous zebrafish, most heterozygous animals develop nor- postfertilization, either spontaneously in homozygotes mally and survive into adulthood. Under normal or following oxidative stress in heterozygotes.29 All conditions, the heterozygous embryos, larvae, or young the retrovirus-mediated insertional mutants showing (adult) fish might not reveal significant vulnerable the altered SA-b-gal activities in homozygous embryos changes immediately in biomarkers of aging as a result or larvae are currently available at the Zebrafish Interna- of the presence of plasticity, such as partial redundant tional Resource Center. compensatory mechanisms and/or adaptive responses It is possible to model hypothetically our mutant and upregulations (eg, hyperactivation) mediated by screening of developmentally essential (potentially the remaining, intact functional allele. However as beneficial vs deleterious) genes for embryonic senes- confirmed by our recent study,29 not only several homo- cence considering the actual aging process (Fig 3). In zygous mutant (complete loss of function) conditions enhancer mutants (harboring mutations in type I genes) but exposures of the corresponding heterozygote car- with increased SA-b-gal activity, the normal allele can riers to specific stress challenges can help to amplify be more beneficial to be against senescence, whereas the response in animals showing dominant, premature the heterozygous allele could be more deleterious in senescence (embryonic senescence) phenotypes even this sense, showing accelerated aging and a subse- during early development (in case of senescence quently shorter life span. (The total 11 mutants Translational Research 128 Kishi February 2014

Fig 3. Modeling of the heterozygote disadvantageous (haploinsufficiency) or advantageous mutation against environmental insults during the aging process. In the upper graph (in mutants of type I genes with a heterozygote disadvantage, or haploinsufficiency), lines shifted to the left (arrows) illustrate a more rapid decline of accuracy in the function of the same gene after early exposure to stress (eg, oxidative stress or gamma radiation, which we used in our studies) in wild-type (Wt) or heterozygous (Het) organisms. According to this model, in wild-type organisms, aging biomarker A manifests around middle age. In heterozygous animals, this biomarker manifests spontaneously, even during early life stages, without stress insult (y-axis) because its threshold lies above the 50% level of gene function, thus predicting an accelerated aging phenotype. In contrast, aging biomarker B is characteristic of old age in wild-type organisms and manifests in heterozygous ones at a relatively earlier period of late adult life. Thus, neither organism would show this biomarker spontaneously during the early stage of life (eg, embryogenesis). However, in both organisms, exposure to stress during development or later in life can change the dynamics of gene function with age, presumably through additional structural and functional damage to cells. As a result, after stress, both aging biomarkers manifest earlier, and aging biomarker B can now be detected earlier in life in the heterozygous organism but not the wild-type one. In homozygous (Homo) mutant (Mu) organisms, either aging biomarker A or B can be detected during early development (ie, increased senescence-associated b-galactosidase [SA-b-gal] activity), and the mutations are lethal early (x-axis). Thus, the search for gene mutations that lead to accelerated aging phenotypes can be conducted during early development of homozygous mutants as well as heterozygous mutants with or without the use of environmental stress factors. Note that although this highly simpli- fied schematic illustrates changes that helped us to predict accelerated aging in zebrafish mutants with altered genes, it does not reflect the more complex relationships between the dynamics of gene functions and age that would be predicted by the antagonistic pleiotropy theory of aging.35,39 Moreover, the concerted effect of numerous genes functioning in parallel throughout life would predictably cause the overall aging process to exhibit nonlinear dynamics, with stochastic environmental factors providing further modifications of age- dependent processes under real-life conditions (eg, epigenetic drift). In the lower graph (in mutants of type II genes with a heterozygote advantage), for heterozygous organisms, aging biomarker A may still manifest a reduction spontaneously during early development without stress insult (y-axis) because its threshold lies above the 50% level of gene function and thus likely predicts any delayed aging phenotype at that point (eg, if decreased SA- b-gal activity can be observed). In contrast, aging biomarker B is characteristic of old age in wild-type animals and manifests itself in heterozygous ones during relatively late adulthood, beyond the age of wild type. Thus, such organisms, which may not show this biomarker spontaneously during early development, but might induce on exposure to stress during development or even later in life, can maintain the robust dynamics of gene function with age, probably as a result of natural resistance to structural and functional damage to cells. As a result, even after stress exposures with age, although aging biomarker A may still manifest a reduction, aging biomarker B can only be detected during middle age of the heterozygous animals beyond the wild-type age. In homozygous mutants, however, aging biomarker A or B could be detected ‘‘negatively’’ during early development (ie, decreased SA-b-gal activity), and these mutations are also lethal early (x-axis). In our experiments, for instance, in the case of enhancer mutants with mutations in type I genes and increased SA-b-gal activity, the normal wild-type allele can be more beneficial to be against senescence, but the heterozygous allele could be more deleterious and can induce accelerated aging and, subsequently, a shorter life span, as seen in the mutants. (The 11 mutants are categorized as type I [Table I].) On the other hand, in suppressor mutants with mutations in type II genes and decreased SA-b-gal Translational Research Volume 163, Number 2 Kishi 129

Table I. Embryonic/larval senescence mutant genes

Embryonic/larval SA-b-gal activity Gene GenBank accession no.

Enhanced by loss of function Spinster homolog 1 (spns1) NM_153663 Telomeric repeat binding factor a (terfa; terf2) NM_173243 Clathrin interactor 1a (clint1a) NM_001003412 ATPase, H1 transporting, lysosomal, V0 subunit c, a (atp6v0ca) NM_001105136 ATPase, H1 transporting, lysosomal, V1 subunit H (atp6v1 h) NM_173270 Polymerase (RNA) II (DNA directed) polypeptide D (polr2 d) NM_001002317 Polymerase (RNA) II (DNA directed) polypeptide G-like (polr2gl) NM_199669 Smoothened homolog (smo) NM_131027 CCR4-NOT transcription complex, subunit 1 (cnot1) NM_001079951 Structural maintenance of chromosomes 1A, like (smc1 al) NM_212810 Denticleless homolog (dtl) NM_173241 Suppressed by loss of function Eukaryotic translation initiation factor 3, subunit D (eif3 d) NM_200016 Ribosomal protein L11 (rpl11) NM_001002139 Small nuclear ribonucleoprotein D1 polypeptide (snrpd1) NM_173252

Abbreviations: ATPase, adenosine triphosphatase; SA-b-gal, senescence-associated b-galactosidase.

categorized in this type I group are presented in Table I.) is intriguing and makes them attractive for further explo- On the other hand, in suppressor mutants (having muta- ration of the possibility of slow or delayed aging regu- tions in type II genes) with decreased SA-b-gal activity, lated by any heterozygote-advantageous condition of the normal allele could be relatively more deleterious in the genotypes. Identification of longevity assurance senescence, but the heterozygous allele may be more genes, which can extend a life span by a mutation, should beneficial against senescence, having a heterozygote be elucidated to establish more fully the existence of the advantage of fitness (the 3 mutants are categorized as physiologically relevant model system for aging in ze- this type II; Table I). These heterozygous organisms brafish. Therefore, we currently anticipate being able would be anticipated to show slow or delayed aging to identify a real suppressor-type aging mutant that dis- and thus a longer life span. plays enhanced stress resistance in advanced age and, Thus, our mutant screen revealed various genotypes perhaps, a longer healthy life span. Alternatively, it designated as both enhancer and suppressor mutants might also be possible to isolate a ‘‘revertant’’ from the with a relative increase and decrease of SA-b-gal background of an accelerated aging mutant, which activity, respectively, during early development. Each would restore the original normal phenotype with reju- gene linked to increase or decreased embryonic SA-b- venation by means of any suppressor mutation (ie, a gal activity is also listed in Table I. We have already reversal of the accelerated aging phenotype). demonstrated that some of enhancer heterozygotes Our studies have demonstrated to date that certain show the accelerated aging phenotypes later in adult environmental challenges (eg, gamma irradiation or life, which underscores the utility of such an approach oxidative stress), which accelerate aging in adult zebra- for identifying early predictors of actual aging phenom- fish, can be effective in revealing biomarkers of aging ena. This suggests that haploinsufficiency in some even during early development. If the coupling of stress genes might not be detrimental to a developing embryo responses in zebrafish embryos and/or larvae with aging under a regular environment, probably as a result of mechanisms in adult fish works in a parallel fashion, as developmental plasticity, but would facilitate alterations has been demonstrated in other invertebrate animal sys- in molecular mechanisms and physiological functions tems and suggested by our results discussed here, this characteristic of aging. If biomarkers of such alterations approach will be a useful and powerful tool in the search could be documented at an early age, they could help to for aging-related genes found more comprehensively in predict subsequent aging phenotypes later in life. vertebrates. Such speedy identification of genes relevant The presence of the lower SA-b-gal levels in the to vertebrate aging using embryos and/or larvae would mutant embryos (suppressor mutants by loss of function) circumvent, in part, the need for time-consuming and

= activity, the normal allele could be more deleterious later in life in terms of senescence, but the heterozygous mutant allele may be more beneficial to be against senescence, having a heterozygote advantage in this regard. (The 3 mutants are categorized as type II [Table I].) These heterozygous animals are anticipated to show slow or delayed aging and a longer life span, whereas the homozygous null mutants have no fitness with lethality. Translational Research 130 Kishi February 2014 lengthy life span analyses in any available zebrafish mu- and pathologies may also originally derive from the tants or even natural variants. drifting actions of developmental programs.51 Age- Although age-related epigenetic alterations remain associated epigenetic drifts in an intrinsic develop- largely to be elucidated in zebrafish, the initial profiles mental program that tends toward imbalance with of decreased cytosine methylation at CpG island shores advancing age can be a cause of the progressive deteri- in some representative genes during zebrafish aging orative changes in physiological functions and homeo- have emerged only recently.18 In this regard, any gene stasis, leading to various diseases.52,53 identified in the zebrafish senescence mutant screening With aging, because regenerative ability also declines would be a highly plausible candidate to be regulated by and degenerative potential increases in zebrafish,15 the both genetic and epigenetic methods under certain envi- loss/decrease of plasticity and/or onset of self-renewal ronmental challenges with actual aging. In particular, defects in stem cells may be responsible for reduced because genetic code mutations occurring in epigenetic regenerative capability and inefficient tissue restoration factors or elements lead to heritable transmission over accompanied by aging. On the other hand, inadequate generations, the transgenerational effect of an epige- elasticity in aging may cause inappropriate regenera- nome still relies significantly on an original genome to tion, potentially leading to cancer or degeneration by some extent. From such a genetic and epigenetic point compromising homeostatic integrity and plasticity. of view, identified genes encoding structural mainte- Of note, the fundamental aging process is not a disease nance of chromosome 1A-like protein and RNA poly- itself, whereas aging obviously increases vulnerability merase II components in our mutant screening must to many diseases, including cancer. In essence, there- be prominent candidates to elaborate the loop or linkage fore, aging may be considered to be one of the most between genomes and epigenomes in terms of an evolu- prominent pathogens and carcinogens. Cancer incidence tionary notion of aging. As such, although we have not increases exponentially with age in most vertebrate spe- specified further any target by means of the canonical cies, as shown in zebrafish as well as in humans.54-56 epigenetic modifications such as DNA methylation, his- This age-associated increase of cancer incidence is pre- tone modifications, and chromatin remodeling, extend- sumably a result of inappropriate developmental drift in ing investigations in epigenetics and epigenomics aging stem cells or aged somatic cells that are trying would guarantee a much deeper understanding of the voluntarily to reset a program having some troubles. complex processes of organismal aging and the mecha- Most of proto-oncogenes function properly in normal nisms of increased risk of disease with age, at the fore- development and growth. Developmentally crucial front of future zebrafish aging research. genes such as oct-3/4 and sox2, and the proto- oncogene c-myc, among others can reprogram differen- EVOLUTIONARY DEVELOPMENTAL ORIGINS OF tiated somatic cells to stem cells beyond plasticity.57-59 AGING AND DISEASE However, such artificially (or even naturally) induced/ Genetic code information determines the architecture reversed ‘‘stemness’’ is insufficiently robust and often of individual organisms during development and pro- suffers from inevitable carcinogenesis because of the grams the mechanisms that maintain adequate functions conflicting relationship between reversing power and extended to adult life, at least to some extent.46 There is forwarding entropy in senescence.60 Inappropriate also substantial evidence that shows that constraints in fading or unexpectedly excessive actions of develop- an early life environment are an important determinant mental functions in adult stages is conceivably caused of the risk of geriatric diseases, including cancer and by less molecular fidelity with increasing entropy.61,62 metabolic disease.47,48 One hypothesis is that the The reason why plasticity is restricted to a particular increased susceptibility to such diseases has a period of early life is, possibly, that there is a difficulty common origin, with developmental changes and in reversing the entire developmental processes of adaptation induced in developing tissues and organs adult organisms in vivo.47 Thus, it might be the result under initially exposed, particular environmental of the unaffordable energy cost of ensuring reproductive conditions, as proposed originally by Barker.49 Devel- success and fidelity in reconstructing the prior, estab- opmental and environmental impacts include nutritional lished characteristics of aging organisms. status and altered epigenetic regulation of any particular Segmental progeroid syndromes, including gene having each genetic background. The induction of Hutchinson-Gilford progeria syndrome (simply referred a subsequent disease risk with age is dependent signifi- to as ‘‘progeria’’), are often accompanied by develop- cantly on the nature of the environmental challenges as mental and growth retardation, but very little is known well as on the interaction of the original genetic suscep- about the developmental process of such accelerated ag- tibility with the altered epigenome throughout the life ing disorders. We have demonstrated the impaired early course.50 However, age-related physiological changes developmental processes of the mesenchymal lineage Translational Research Volume 163, Number 2 Kishi 131 in zebrafish embryos harboring disturbed lamin A presumably because premature aging during early life expression,63 and other lines of evidence also support has often been considered pathologic, rather than phys- the notion that developmental signaling pathways are iological, as a canonical context. Conceivably, howev- presumably involved in lamin A-associated aging mech- er, our identified genes of senescence enhancers by loss anisms as well.64 Because embryonic stem cells appear to of function might encode juvenile protective and/or express lamin A/C on differentiation,65,66 and lamin rejuvenating factors that could act normally to delay A-associated stem cell dysfunction has been linked to aging and that, when mutated, can cause premature disruption of the Wnt and Notch signaling path- aging. ways,64,67-69 which are essential for early vertebrate The morphogenic signals mediated by Hedgehog and development, our observations of embryonic senes- Wnt are essential to direct pattern formation during cence or apoptosis in lamin A gene (LMNA)-disturbed embryogenesis, and both the signal activities can remain zebrafish might be consistent with a dysregulation of through adulthood. Consistently, it has been reported somatic stem cells leading to organismal premature that both Hedgehog and Wnt pathways function in ag- aging. In particular, it is plausible that the less plasticity ing.80-83 In addition, critical roles of Hedgehog and and insufficient self-renewal ability in mesenchymal Wnt signals have been implicated in metabolic stem cells may be responsible for the specificity of accel- regulatory events, including insulin sensitivity and fat erated tissue degeneration, as most affected tissues in formation.84,85 In lines with these, as we described, our progeria are of mesenchymal origin.68 Lamin A has embryonic senescence mutant screens have identified also emerged recently as a key player vulnerable to the Hedgehog signal transducer, Smoothened (Smo), epigenetic changes that contribute to aging.70-73 as a driver of SA-b-gal activity by the loss of its Embryonic senescence and further unforeseen develop- function.29 Moreover, haploinsufficient, accelerated ag- mental abnormalities may also be occurring in human ing phenotypes in retinal photoreceptors via the hetero- (or mice) progeria in either de novo genetic and/or zygous hedgehog genotype have been reported in epigenetic manners, given that severe neonatal lethality zebrafish.80 Thus, Hedgehog signaling may function as has been reported.74-76 Thus, aging research should an aging antagonist or rejuvenating factor.86,87 The therefore extend to the study of early developmental outcome of several plausible candidate mutants in our processes at least within the context of reproduction mutant screening, such as smo mutant fish, has and regeneration. provided a proof of concept for our original approach It is reasonable to assume that the developmental the- to assess the rejuvenating connection between develop- ory of aging approach leads to the identification of ge- mental and senescence signaling pathways through netic and epigenetic pathways that are relevant for common genes during embryogenesis in zebrafish. development alone, for aging alone, and for both devel- The secreted factor Klotho, which acts as a proficient opment and aging.77 Subsequently, it would be possible suppressor of aging, may also be classified as a rejuve- to establish a concept and experimental evidence of the nating factor. Klotho-deficient mice manifest a syndrome spiral loop of genome and epigenome that can be linked resembling accelerated human aging with multiple dete- to organismal evolution in aging and disease. As a riorative features,88 whereas Klotho-overexpressed mice fundamental task at this stage, the pathways and related have been found to have an extended life span that dis- molecules that have already been implicated in aging plays antiaging properties.89 In Klotho-deficient animals, and longevity need to be investigated more extensively increased Wnt signaling activity becomes evident; in in early development, as characterized for the several fact, Klotho functions as a secreted Wnt antagonist. gene products, including lamin A, ataxia telangiectasia Thus, Klotho deficiency-induced accelerated aging is a mutated, and telomerase.63,78,79 result, in part, of constitutive activation of Wnt signaling. Hence, an unexpected deleterious role of Wnt in aging REJUVENATING FACTORS AGAINST AGING AND has been revealed in a Klotho-deficient mouse model sys- DISEASE tem, whereas other lines of evidence support contradicto- Embryos, larvae, and juveniles of many species have rily beneficial effects of a Wnt signaling against aging a greater potential for tissue repair and regeneration because it can ameliorate multiple age-associated symp- than adults, and eventually they lose such ability with toms.90 A tissue-dependent imbalanced regulation age, in parallel with more disease. Somehow, little between Klotho and Wnt may be caused by develop- attention has been devoted to determining physiolog- mental drift in aging stem cells and may be a reason for ical factors characteristic of early life that could reduced plasticity in tissues and organs with age. contribute to healthy aging and a healthy life span pro- More important, as well, essential involvement of the tective against adult-onset/geriatric diseases. This is Notch and Wnt signaling pathways in the deterioration Translational Research 132 Kishi February 2014 of stem cells from patients with progeria and model mice study of aging phenotypes among the zebrafish mutants have been described, respectively.64,67-69 In human that were identified based on early-life evaluations. This progeria, Notch signaling is upregulated in and other examples indicate that zebrafish can be an mesenchymal stem cells, leading to activation of the ideal system to develop alternative vertebrate models downstream target genes and to increased osteogenesis to address the many biologic questions regarding the and decreased adipogenesis by perturbing cellular relationship between development and aging from differentiation. Furthermore, in progeria model mice, both genetic and epigenetic points of view, in the most the transcriptionally active form of b-catenin, which is amenable throughput setting. the well-characterized regulator of the canonical Wnt Aging has been thought of as a stochastically deteri- signaling pathway, was almost completely absent in orative and destructive process, whereas development hair follicle stem cells. is a finely programmed and constructive process. How- Moreover, given the recent findings that Wnt signaling ever, the genetic and epigenetic robustness of aging may and the telomerase catalytic subunit (telomerase reverse already be defined during early developmental stages to transcriptase [TERT]) can potentially make a positive a certain extent. Thus, a certain gene mutation might regulatory loop—the Wnt pathway component b-cate- lead to accelerated decline in gene function that would nin can regulate positively the expression of TERT normally occur only at old age with a particular whereas TERT can regulate positively the expression threshold and/or window, and this process may be facil- of Wnt/b-catenin target genes91,92—it is now itated or modulated by the challenge of multiple reasonable to propose that the new direct link between stressors that can mimic various environmental impacts. these mechanistic regulations has a crucial impact on In such cases, dependable biomarkers of aging could be balancing regeneration/rejuvenation and cancer during revealed, potentially, at a much earlier age. As we the aging process beyond embryogenesis.93 have shown in a previous study, this issue can be Intriguingly, a recent study revealed that the cardiac explored fruitfully using unbiased mutant screens for hypertrophy of aging was, at least in part, mediated by senescence-associated biomarkers during early devel- circulating factors, and that systemic growth differenti- opmental stages.29 Overall, I propose that the ation factor 11, a transforming growth factor b family complexity of aging in higher organisms, involving member, could reverse age-related cardiac hypertrophy. the interaction of multiple genes and signaling path- This finding suggests that at least 1 pathologic compo- ways, warrants a comprehensive search for evolutionary nent of age-related diastolic heart failure is hormonal developmental origins as early predictors of altered ag- in nature and reversible by treatment with such a rejuve- ing phenotypes later in life, when we might be able to nating factor.94 reverse the process by rejuvenation. The zebrafish ani- Taken together, the cumulative lines of evidence sup- mal model, with its well-established genetics and port the notion that developmental signaling pathways advanced high-throughput technologies, offers an and their possible drifting actions may be involved in unparalleled opportunity to identify aging-related ge- aging and rejuvenation mechanisms,51 and we recog- netic and epigenetic elements, and to analyze their func- nize that the zebrafish model system can contribute tion easily throughout the life span. The highly enormously to the identification of rejuvenating factors conserved or even unique mechanisms of aging at the against aging and the study of molecular mechanisms to cellular, tissue, and organismal levels can and should reverse the aging process that are common among ver- be addressed using zebrafish as the most affordable ge- tebrates. netic and epigenetic model in vertebrates, comparable with C. elegans and Drosophila in invertebrates.95,96

CONCLUSIONS The author is grateful to the current and former members of my laboratory. He also thanks Yi (Eric) Shi for help with the illustrations. Our findings from recent studies suggest that embryonic senescence in zebrafish can be connected to the REFERENCES process of actual aging, which manifests at both the 1. Hasty P, Campisi J, Hoeijmakers J, van Steeg H, Vijg J. Aging and genome maintenance: lessons from the mouse? Science 2003; cellular and organismal levels. These include morpho- 299:1355–9. logic and histologic changes in multiple tissues, reduced 2. Seifert AW, Voss SR. Revisiting the relationship between regener- regeneration, and damage to macromolecules (DNA, ative ability and aging. BMC Biol 2013;11:2. proteins, and lipids), lipofuscin accumulation possibly 3. Carey JR, Liedo P, Harshman L, et al. Life history response of resulting in reduced intracellular trafficking, cognitive Mediterranean fruit flies to dietary restriction. Aging Cell 2002; 1:140–8. decline, circadian dysregulation, and sleep alterations, 4. Liao CY, Rikke BA, Johnson TE, Diaz V, Nelson JF. Genetic vari- among others. These hallmarks of aging, also typical ation in the murine life span response to dietary restriction: from of other vertebrates, have been used in our longitudinal life extension to life shortening. Aging Cell 2010;9:92–5. Translational Research Volume 163, Number 2 Kishi 133

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