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The African turquoise killifish Nothobranchius furzeri as a model for aging research
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. 27, 2018
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
Models for Aging Research
The African turquoise killifish Nothobranchius furzeri as a model for aging research Hanna Reutera,1, Johannes Kruga,1, Peter Singera,1, Christoph Englerta,b,* a
Molecular Genetics Lab, Leibniz Institute on Aging–Fritz Lipmann Institute (FLI), Beutenbergstr. 11, 07745 Jena, Germany Institute of Biochemistry and Biophysics, Friedrich-Schiller-University Jena, Hans-Knöll-Str. 2, 07745 Jena, Germany
b
Nothobranchius furzeri is an African killifish with an exceptionally short median life span of 3 to 7 months. Despite its short life span N. furzeri displays signs of
Section editor: Dr. Hildegard Mack – Institute for Biomedical Aging Research, University of Innsbruck, Austria.
aging that are highly reminiscent of mammalian aging. In 2015, reference sequences for the N. furzeri genome have been published. In addition, transgenesis and genomic engineering of killifish using CRISPR/Cas9 have been recently established. It has also been shown that N. furzeri is amenable to pharmacologic intervention. In this review we will discuss what makes N. furzeri a valuable model for research on aging and whether it could also be used for drug discovery.
Introduction With the rise in life expectancy research on aging and agingassociated diseases is drawing increasing attention. In fact, while many disciplines within the biomedical sciences have sporadically considered aspects of aging, aging research has meanwhile become a discipline of its own. This becomes evident by the foundation of a number of institutes that are dedicated exclusively to research on aging. This process has started with the Buck Institute for Research on Aging in Novato, California and was followed by a number of insti*Corresponding author: C. Englert (Christoph.englert@leibniz-fli.de) 1 These authors contributed equally to the manuscript.
tutes in the US, Europe and Asia. Traditionally, the main model organisms in research on aging are yeast, Caenorhabditis elegans, Drosophila and the mouse. Recently, these wellestablished models have been joined by exceptionally longas well as short-lived vertebrates as new aging models, namely the naked mole rat [1] and the African killifish Nothobranchius furzeri [2,3]. The latter lives in seasonal ponds in southeast Africa and is the shortest-lived vertebrate that can be kept in captivity [3]. Depending on the strain N. furzeri has a median life span between 3 and 7 months and displays rapid growth and early sexual maturation [4,5]. What makes N. furzeri particularly appealing is established transgenesis [6–8] and genome editing [9] and the recent publication of a reference sequence for its genome [10,11]. In this review we discuss why N. furzeri is a valuable model for research and consider how killifish might be used for discovery and characterization of drugs that decelerate the aging process.
N. furzeri: life cycle and life span With less than one year of maximum life span, N. furzeri is an ideal model for research on aging. The short life span is
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considered to be an adaptation to its ephemeral natural habitat in South-Eastern Africa with alternating rainy and dry seasons. The fertilized killifish eggs and developing embryos survive extended dry seasons in an arrested state, called diapause that can occur at three different phases of embryonic development. To date, we still know very little about the mechanisms that trigger onset and duration of each diapause, however, low temperature and low oxygen seem to favor the onset of diapause, at least in N. guentheri [12]. When the rainy season begins, the embryos hatch and the larvae rapidly grow to sexual maturity (Fig. 1A,C), which can be reached as early as 14 days after hatching [5]. Currently, several N. furzeri strains that differ in origin and life span are used for biomedical research. The GRZ strain was originally isolated in 1969 in a semi-arid habitat in Zimbabwe, has a maximum life span of 4–6 months and is highly inbred [3,13,14]. Strains from more humid regions in Mozambique were established only several years ago, are genetically heterogeneous and have a longer maximum life span of 40–60 weeks [15,16]. Thus, the life span of N. furzeri is exceptionally short compared to other fish species that are used to model human diseases and aging like zebrafish or medaka with median life spans of 60 and 22 months, respectively [17,18]. In a very recent field study it was shown that the fast growth and rapid sexual maturation of N. furzeri that is observed in captivity is probably an underestimate of the timing of the natural processes. Natural populations that were surveyed by measuring otolith growth and gonad histology grew within two weeks from 5 to 54 mm and reached sexual maturity after 14 days [5]. Most likely, this is the fastest rate of sexual maturation in a vertebrate. The availability of natural populations and laboratory strains that differ largely in life span has enabled a quantitative trait locus (QTL) analysis regarding loci that determine life span. To this end a short- and a long-lived strain were crossed, life span was recorded and a QTL analysis was performed [19]. By combining genotype and phenotype information, four life span QTLs which taken together explain 27% of the total life span variance in the F2 generation were identified. The genes that are contained within the respective genomic regions can be considered as candidate genes for the determination of vertebrate life span.
dase and lipofuscin as well as the cell cycle inhibitors p21 and p16 have been shown to accumulate during N. furzeri aging [24,25]. Again, these markers are found in all other vertebrate aging models and are present in humans [26]. On an organismic level, aging in N. furzeri manifests itself by reduced coloration in males, malformations of the spine and face, and weight loss (Fig. 1B). These phenotypic changes are paralleled by changes in behavior, e.g. locomotor activity, open-field exploration as well as learning and memory function [24,27,28]. In addition, an age-dependent decay in adult neurogenesis has been described for N. furzeri [29]. This suggests aging-associated exhaustion of neuronal stem cells, which is another important hallmark of aging. Loss of regenerative capacity has also been observe with regard to the N. furzeri caudal fin [16]. Other hallmarks of aging have recently been proposed, including loss of proteostasis, epigenetic alterations and altered intercellular communication [26]. While direct evidence for some of those has not yet been obtained in N. furzeri, there is no reason to believe that they are absent or cannot be studied in killifish. Perhaps surprisingly, N. furzeri displays a high incidence of tumors, particularly in the liver and the kidney [30]. It should be noted, however, that in fish the kidney is the main hematopoietic organ. Therefore, it is likely that tumors in the kidney are leukemias rather than tumors of the kidney itself. It is still unexpected to find a high cancer rate in such a short-lived organism and suggests that killifish might also serve as models for tumorigenesis. Finally, the short life span of N. furzeri makes longitudinal studies feasible. In a first study of such kind gene expression during early life was correlated with individual life span [31]. One of the key results of this study was that expression levels of several gene sets, including oxidative phosphorylation, showed lower expression levels early in the life of long-lived individuals. This raised the hypothesis that expression of genes encoding components of the respiratory chain might be negatively correlated with individual life span. It was indeed shown in a subsequent experiment that treatment of N. furzeri with a low dose of rotenone, an inhibitor of complex I reverted gene expression signatures to those of younger age and led to extension of life span by 15%.
Hallmarks of aging in N. furzeri
Degeneration and regeneration
Despite its short life span N. furzeri shows many hallmarks that are typical signs of mammalian aging at the molecular, cellular, organ as well as organismic level including behavior. Telomere shortening is one of those hallmarks that have been reported to accompany aging in N. furzeri [20]. This is particularly interesting as in terms of length killifish telomeres are much more similar to human telomeres than the much longer mouse telomeres [21,22]. Also, mitochondrial dysfunction has been reported to occur in killifish upon aging [23]. In terms of molecular senescence markers, b-galactosi-
Repair processes are limited to a few organ systems in humans. Importantly, processes linked to regeneration such as wound healing often decline with age. The rate of reepithelialization after wounding is strongly affected in old humans [32] and children display better regrowth of lost fingertips than adults [33]. Regenerative therapies are a promising approach to reverse aging damage and restore functionality in the elderly. Degeneration and respective regeneration processes are complex, involving the interplay of many different cell types and factors that are difficult to fully model in
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telomere shortening mitochondrial dysfunction increase in aging markers (lipofuscin, senescence) oxidative stress (heart) neurodegeneration decreased regeneration (fin) Drug Discovery Today: Disease Models
Fig. 1. Rapid growth and aging in N. furzeri. (A) Rapid growth of N. furzeri GRZ (median life span of 3–7 months). For fish breeding, the dry/wet season conditions are mimicked in the lab whereby the fertilized N. furzeri eggs are collected on peat moss plates. While the hatchling is only a few millimeters of size, the adult fish reach a size of several centimeters. The colorful males are bigger in size than the grey female fish. Scale bar is 1 cm. (B) Representative pictures of young, adult and old male fish illustrate phenotypical changes with age (MZSC-08/122 strain that has a mean life span of about 11 months). Signs of aging on a phenotypical as well as molecular level are listed. Scale bar is 1 cm. (C) Schematic illustration of the rapid increase in body size with age.
vitro. The need of animal models to understand such processes is therefore of great importance. In contrast to humans, fish have been reported to have extraordinary regeneration capacities. The most prominent example is zebrafish that is
known to repair complex organs such as the brain, spinal cord, kidney, heart and appendages after severe injury (reviewed in Ref. [34]). Regeneration capacities of the caudal fin as well as the kidney have been reported for N. furzeri www.drugdiscoverytoday.com
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[16,35]. Functionality of the killifish kidney is restored faster after damage than in zebrafish: reabsorption of a fluorescently labeled sugar is almost back to normal already 8 days post injury, while kidney function recovery is only observed 2–3 weeks post injury in D. rerio [35–37]. Apoptotic levels peaked in renal tubules 2 days post injury in N. furzeri, which was accompanied by an increase of proliferating cells in tubules. Newly developing nephrons (neonephrogenesis) could already be observed 4 days post damage. This study suggests that tubular regeneration allows fast functional recovery in N. furzeri and is followed by neonephrogenesis [35]. Whereas repair of the damaged kidney by surviving tubular epithelial cells is common to mammals as well [38], neonephrogenesis seems to be distinctive to teleost fish. N. furzeri therefore offers the great opportunity to study molecular processes underlying the formation of new tubules after kidney damage, especially in an aging context. Caudal fins of young adult N. furzeri are nearly completely regenerated within 27 days after amputation, which is similar to the restoration of fin shape and size after 3 weeks in zebrafish [39]. Interestingly, an age-dependent decline in fin regenerative capacity has been reported in N. furzeri where fish with a mean age of 54 weeks (long-lived MZM-0703 strain) only regenerated 46% of their original fin size [16]. However, the observation that young fish regenerate faster than old fish has already been noted in Pierre M.A. Broussonet’s first description of fin regeneration in the 18th century when he experimented on goldfish [40]. For zebrafish, there are conflicting reports regarding agedependency. While one study reported life-long regeneration capacities for the zebrafish fin [41], other studies suggest that old animals show impaired fin regeneration [42,43]. A general drawback of the zebrafish compared to the killifish studies is a less careful definition of age groups. This might be a possible reason for opposing results. Zebrafish over 24 months of age are often considered as ‘old fish’ although they can live up to 5– 6 years. The short life span and the clear results concerning agedependent decline in regeneration capacities make N. furzeri the more powerful model system to study age-dependent regeneration, especially since animals from the same clutch can be more easily studied over time. For the killifish fin, molecular differences concerning age-dependency have been identified, whereas the zebrafish studies that reported agedifferences mainly compared macroscopic differences. Early key events during regeneration in old and young animals were identified to be different in killifish fins post amputation. Old fish showed a delay in bone formation, proliferation levels were higher in young fish fins while apoptotic cell numbers were increased in the regenerating fin of old fish [16]. Expression analysis of genes involved in fin regeneration showed an increased expression post amputation in all age groups studied, but older fish show a delay in peak expression of several genes (shh, fgf20a, fgfr1, wnt10a, raldh2, lef1 and msxb) [16]. 18
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The study that reported life-long regeneration capacities for the zebrafish fin also suggests the same for the zebrafish heart [41]. Since heart failure is an important health issue in elderly humans, it would be interesting to have an agedependent regeneration model for the heart as well. The neonatal mouse heart possesses a transient regenerative potential but the adult mouse heart lacks such regenerative capacities [44]. The human heart fails to regenerate after injury but cardiomyocyte renewal has been observed in young humans, although at low rate, while it is almost neglectable in adults [45,46]. With regard to heart failure and replacement of damaged heart muscle, this observation of cardiomyocyte proliferation in young humans is promising for the development of regenerative strategies in elderly. Whether the killifish heart is an appropriate system to study the aging heart remains elusive. The killifish heart is still poorly studied. Post-mortem analyses indicated an aging phenotype such as hypertrophy and heart dilation [30]. A study comparing old and young killifish hearts reported an increase of oxidative stress and changes in cardiac miRNA expression with age in killifish hearts [47]. It might be questionable whether the killifish heart responds similarly to damage as the zebrafish heart since the closer relative medaka shows impaired heart regeneration [48,49]. The nervous system is another important aspect concerning degeneration and regeneration processes. Neurodegeneration such as neurofibrillary degeneration and accumulation of the age-related pigment lipofuscin in the brain have been reported for N. furzeri [27,50]. This observed neurodegeneration is in line with decay in cognitive function assessed by open field exploration and learning performance in a shuttle box [27,50,51]. Interestingly, dietary restriction was able to reduce neurodegeneration and helped to improve learning performance [52]. This observation indicates that N. furzeri is a promising model to find tools to stop degeneration and restore regeneration capacities in the elderly. More organ systems in N. furzeri need to be investigated for their regeneration capacity in future to expand models for human degenerative diseases. In order to understand how regenerative strategies can potentially be applied in humans to fight aging damage, it is of special interest to study regeneration processes in an age-related context. Only by using such a model system that allows us to combine regeneration and age research, we will be able to elucidate which cellular and molecular processes underlie regeneration in general and the observed decline in regenerative capacities with age.
Genome engineering in N. furzeri Gene inactivation is state-of-the-art to study gene function in vivo. The classical forward genetics approach is based on the random generation of mutations followed by the subsequent analysis identifying the genetic alteration causing the observed phenotype. Those random mutations were induced by
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genome sequence can be found in the N. furzeri Genome Browser (NFINgb) as well as the African Turquoise Killifish Genome Browser. Further access to sequence and transcriptome data is possible via BLAST and the N. furzeri transcriptome browser (NFINtb) allowing for comparisons with other species. All target-specific genome-editing systems are based on the induction of a DNA double-strand break in the locus of interest stimulating the DNA damage response pathways. Those double strand breaks can either be repaired via the error-prone non-homologous end joining (NHEJ) or the highfidelity homology-directed repair (HDR) mechanism [59]. The introduction of the CRISPR/Cas9 technology in 2012 started to revolutionize the field of sequence-specific genome-editing, since it is cheaper and easier applicable than the earlier employed zinc-finger nucleases (ZNFs) and transcription activator-like effector nucleases (TALENs) [60]. Naturally, the CRISPR/Cas system is the adaptive immune system in microbes, which recognizes and degrades foreign nucleic acids from viruses and bacteriophages [61]. Since its identification as a tool for genomic engineering, the CRIPSR/Cas technology has been widely applied in a broad range of model organisms starting with human and mouse cells [62], Danio rerio [63] and Mus musculus [64]. To achieve successful genome-engineering, sequence-specific single guide (sg)RNAs are guiding the Cas9 endonuclease to the sequence of interest, where a DSB is induced. In 2015, the first CRISPR/Casinduced knockouts were described in N. furzeri [9] (Fig. 2). Since N. furzeri has been established primarily as a model organism for aging research, the targeted genes were all linked to aging (e.g. igf1r, tert) in order to study the aging
the exposure to mutagens such as X-rays [53] or chemicals like ethyl methanesulfonate [54]. The nowadays commonly used reverse genetics techniques are used to firstly induce mutations in the gene of interest and then study its impact on the organism. However, this approach requires two main prerequisites: a sequence-specific genome editing tool as well as an assembled and annotated genome. With the help of the first genetic linkage map based on microsatellites [55] and a partial sequence of the N. furzeri genome obtained by Sanger sequencing [14] the first genetic modifications were possible. In 2011, a first protocol for transposon-mediated transgenesis in N. furzeri was published [8] (Fig. 2). This transgenesis approach is based on the Tol2 transposon system, which originates from Oryzias latipes (Medaka) [56], and has previously also been successfully applied in other model organisms such as zebrafish [57] and mouse [58]. With this system a construct flanked with Tol2 recognition sites can be inserted randomly into the genome. Due to the thick chorion surrounding the N. furzeri egg, microinjection protocols had to be adapted in order to get access to the cell for the injection [7,8]. Based on those protocols the first transgenic N. furzeri lines expressing GFP under the control of the Cska or Hsp70 promoter were generated, allowing ubiquitous as well as inducible expression of transgenes such as a heat-shock induced reversible expression of GFP [6,8] (Fig. 2). Tools for sequence-specific gene modifications have been established during the last years and were already frequently applied in various model organisms. Together with the publication of the N. furzeri (1.24 Gb) genome in 2015 [10,11], this was a milestone for sequence-specific genome engineering in N. furzeri (Fig. 2). The N. furzeri
Nothobranchius furzeri as model for aging research
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Fig. 2. Hallmarks in Nothobranchius furzeri research. During the last 15 years, N. furzeri has been established as a model organism for aging research. The scheme summarizes important hallmarks in the research on the short-lived African killifish. Pictures adapted from Ref. [7]. www.drugdiscoverytoday.com
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process and age-associated diseases. As an example a telomerase-deficient tert knockout line was generated to analyze telomere attrition during aging. Besides the short life span N. furzeri has telomeres of a similar size range as humans, in contrast to mice that have to be bred for several generations until the occurrence of telomerase-related phenotypes [9]. Besides the knockout of several genes, knock-in experiments using the CRISPR/Cas9 system in the short-lived killifish were also performed [9,65]. Instead of the error-prone NHEJ, the precise homology-directed repair (HDR) mechanism is needed to repair the Cas9-induced double-strand break. For this purpose a single-strand DNA template needs to be provided that can then be inserted at the desired region of interest. This HDR-based engineering can be used to insert exogenous DNA sequences or precisely alter genomic sequences at specific target sites. By this, specific human disease-causing mutations of tert have been introduced in N. furzeri [9]. Moreover, this technique could be applied to insert for example loxPsites or reporter genes into specific loci to further expand the experimental applications of N. furzeri. The short life span of N. furzeri comes along with a rapid growth and an early sexual maturity after only two to three weeks in the short-lived strain [4,5]. This allows a rapid generation of stable genetically altered N. furzeri lines within two to three months [9,65]. In contrast, for the generation of a stable D. rerio line, a time span of 2.5–4 months is already needed to reach sexual maturity in F0 fish [66]. Despite its rapid growth, the embryonic development of N. furzeri is very slow [67]. The one-cell stage in N. furzeri, used for microinjections, can last up to a few hours, greatly increasing the chances of an early induction of the genomic modification and thereby reducing mosaicism. Both, the availability of an annotated genome as well as established genomic modification protocols make N. furzeri a suitable model organism for various biological research fields. Genomic alterations in N. furzeri are currently mainly focusing on aging and aging-related disease. Additionally, specific modifications of receptors, genes involved in regulatory pathways and others can be used to study impacts and effects of specific drugs.
Influences on life span by environmental conditions and drugs In recent years two different strategies have been employed to delay aging and the onset of aging-associated diseases, namely genetic and pharmacological approaches [68,69]. Of late, senolytic drugs that can selectively eliminate senescent cells have come into focus [70]. In this regard, the question arises whether N. furzeri might be an appropriate model for drug testing. The drug resveratrol, which has been shown to retard aging in a number of aging models like yeast, worm and fly, has indeed been shown to prolong life span and delay the onset of aging markers in N. furzeri [50]. Another intervention 20
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that has been shown to increase life span in N. furzeri was reducing the water temperature [28]. Also, the effect of dietary restriction (DR) on aging and longevity has been tested in two different lines, namely an inbred and a longer-lived wildderived line of N. furzeri [52]. While DR prevented the occurrence of the age marker lipofuscin in the brain and improved performance in a learning test there were differential effects regarding life span. Whereas in the inbred strain overall agerelated risk of mortality was reduced, in the wild-derived strain DR even induced early mortality. In both lines DR extended maximum life span, albeit to different degrees. In terms of drugs, the only other substance that had been administered to N. furzeri to date is rotenone, an inhibitor of mitochondrial respiratory chain complex I [31]. Rotenone extended life span by 15% and reversed aging-associated gene expression profiles in different organs toward younger age. This effect is most likely a hormetic response, as a higher dose of rotenone in N. furzeri was life-shortening [31]. Of note, killifish has recently also been used for ecotoxicological testing regarding for example the effects of cadmium and copper [71,72]. Although reports on pharmacological screens are still scarce, the application of resveratrol and rotenone on N. furzeri show that this species is well-suited for the identification of pharmacological modifiers of aging and regeneration. Because of its territorial behavior, particularly male N. furzeri animals are often kept in single housing tanks. Also, most killifish facilities operate with a system in which multiple tanks are connected. This limits the possibility to perform large-scale pharmacological screens. However, the accessibility of several aging markers enables small- and mid-scale pharmacological screens with the goal to identify drugs, which delay aging and the onset of aging-related diseases. The killifish model is therefore best suited as a secondary screening model in which a certain number of candidate drugs that have been obtained by a primary screen (e.g. in cell culture) shall be validated in a vertebrate model.
Outlook A promising approach to find interventions for aging in humans and to prolong health span is to combine the described genomic editing, regeneration research and drug screening approaches described in this review. On this route, N. furzeri with several natural strains of different life span, the possibility to edit its genome, the availability of its genome sequence and the possibility to perform genetic and pharmacological screens for genes and substances affecting aging and longevity is emerging as a very promising model. Recently, the microbiota, particularly the gut microbiota has been shown to influence multiple processes in vertebrates including aging [73]. Also in this regard, N. furzeri turns out to be a very useful model [74]. It was shown that transferring the gut microbiota from young to middle-aged individuals prevented behavioral decline and extended life span.
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Finally, to further increase the acceptance of N. furzeri as a model for different research fields, it is necessary to establish general housing and breeding conditions for this species as has been suggested earlier [75]. Additionally, current efforts to develop a standardized diet aim to minimize the risk of contamination caused by the widely used live food. The Nothobranchius Symposium, held every two years, serves as a platform for the exchange of knowledge and improvements concerning research and husbandry.
Acknowledgements We want to thank all members of the Nothobranchius project at FLI for their contributions and particularly thank the fish facility for excellent fish care over the years. The authors also acknowledge continuous support from the Leibniz Association, the German Federal Ministry of Education and Research (BMBF), and the German Research Foundation (DFG).
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[17] Ding L, Kuhne WW, Hinton DE, Song J, Dynan WS. Quantifiable biomarkers of normal aging in the Japanese medaka fish (Oryzias latipes). PLoS One 2010;5(10)e13287. [18] Lakhina V, Murphy CT. Genome sequencing fishes out longevity genes. Cell 2015;163(6):1312–3. [19] Kirschner J, Weber D, Neuschl C, Franke A, Bottger M, Zielke L, et al. Mapping of quantitative trait loci controlling lifespan in the short-lived fish Nothobranchius furzeri–a new vertebrate model for age research. Aging Cell 2012;11(2):252–61. [20] Hartmann N, Reichwald K, Lechel A, Graf M, Kirschner J, Dorn A, et al. Telomeres shorten while Tert expression increases during ageing of the short-lived fish Nothobranchius furzeri. Mech Ageing Dev 2009;130(5):290–6. [21] Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345(6274):458–60. [22] Kipling D, Cooke HJ. Hypervariable ultra-long telomeres in mice. Nature 1990;347(6291):400–2. [23] Hartmann N, Reichwald K, Wittig I, Drose S, Schmeisser S, Luck C, et al. Mitochondrial DNA copy number and function decrease with age in the short-lived fish Nothobranchius furzeri. Aging Cell 2011;10(5):824–31. [24] 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(5):223–33. [25] 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(1):17–28. [26] Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell 2013;153(6):1194–217. [27] Terzibasi E, Valenzano DR, Benedetti M, Roncaglia P, Cattaneo A, Domenici L, et al. Large differences in aging phenotype between strains of the shortlived annual fish Nothobranchius furzeri. PLoS One 2008;3(12)e3866. [28] Valenzano DR, Terzibasi E, Cattaneo A, Domenici L, Cellerino A. Temperature affects longevity and age-related locomotor and cognitive decay in the shortlived fish Nothobranchius furzeri. Aging Cell 2006;5(3):275–8. [29] Tozzini ET, Baumgart M, Battistoni G, Cellerino A. Adult neurogenesis in the short-lived teleost Nothobranchius furzeri: localization of neurogenic niches, molecular characterization and effects of aging. Aging Cell 2012;11(2):241–51. [30] Di Cicco E, Tozzini ET, Rossi G, Cellerino A. The short-lived annual fish Nothobranchius furzeri shows a typical teleost aging process reinforced by high incidence of age-dependent neoplasias. Exp Gerontol 2011;46(4):249– 56. [31] Baumgart M, Priebe S, Groth M, Hartmann N, Menzel U, Pandolfini L, et al. Longitudinal RNA-seq analysis of vertebrate aging identifies mitochondrial complex I as a small-molecule-sensitive modifier of lifespan. Cell Syst 2016;2:1–11. [32] Ashcroft GS, Dodsworth J, van Boxtel E, Tarnuzzer RW, Horan MA, Schultz GS, et al. Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels. Nat Med 1997;3(11):1209–15. [33] Illingworth CM. Trapped fingers and amputated finger tips in children. J Pediatr Surg 1974;9(6):853–8. [34] Gemberling M, Bailey TJ, Hyde DR, Poss KD. The zebrafish as a model for complex tissue regeneration. Trends Genet 2013;29(11):611–20. [35] Hoppe B, Pietsch S, Franke M, Engel S, Groth M, Platzer M, et al. MiR-21 is required for efficient kidney regeneration in fish. BMC Dev Biol 2015;15:43. [36] Diep CQ, Ma D, Deo RC, Holm TM, Naylor RW, Arora N, et al. Identification of adult nephron progenitors capable of kidney regeneration in zebrafish. Nature 2011;470(7332):95–100. [37] McCampbell KK, Springer KN, Wingert RA. Atlas of Cellular Dynamics during Zebrafish adult kidney regeneration. Stem Cells Int 2015;547636. [38] Humphreys BD, Valerius MT, Kobayashi A, Mugford JW, Soeung S, Duffield JS, et al. Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell 2008;2(3):284–91. [39] Pfefferli C, Jazwinska A. The art of fin regeneration in zebrafish. Regeneration (Oxf) 2015;2(2):72–83. [40] Broussonet M. Observations sur la re´ge´ne´ration de quelques parties du corps des poissons. In: Histoire de l’Acade´mie Royale des Sciences. 1786;p.
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[41] Itou J, Kawakami H, Burgoyne T, Kawakami Y. Life-long preservation of the regenerative capacity in the fin and heart in zebrafish. Biol Open 2012;1(8):739–46. [42] 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(2): 209–224. [43] Anchelin M, Murcia L, Alcaraz-Perez F, Garcia-Navarro EM, Cayuela ML. Behaviour of telomere and telomerase during aging and regeneration in zebrafish. PLoS One 2011;6(2)e16955. [44] Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, Olson EN, et al. Transient regenerative potential of the neonatal mouse heart. Science 2011;331(6020):1078–80. [45] Mollova M, Bersell K, Walsh S, Savla J, Das LT, Park SY, et al. Cardiomyocyte proliferation contributes to heart growth in young humans. Proc Natl Acad Sci U S A 2013;110(4):1446–51. [46] Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabe-Heider F, Walsh S, et al. Evidence for cardiomyocyte renewal in humans. Science 2009;324 (5923):98–102. [47] Heid J, Cencioni C, Ripa R, Baumgart M, Atlante S, Milano G, et al. Agedependent increase of oxidative stress regulates microRNA-29 family preserving cardiac health. Sci Rep 2017;7(1):16839. [48] Ito K, Morioka M, Kimura S, Tasaki M, Inohaya K, Kudo A. Differential reparative phenotypes between zebrafish and medaka after cardiac injury. Dev Dyn 2014;243(9):1106–15. [49] Lai SL, Marin-Juez R, Moura PL, Kuenne C, Lai JKH, Tsedeke AT, et al. Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration. Elife 2017;6. [50] Valenzano DR, Terzibasi E, Genade T, Cattaneo A, Domenici L, Cellerino A. Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr Biol 2006;16(3):296–300. [51] Valenzano DR, Cellerino A. Resveratrol and the pharmacology of aging: a new vertebrate model to validate an old molecule. Cell Cycle 2006;5 (10):1027–32. [52] Terzibasi E, Lefrancois C, Domenici P, Hartmann N, Graf M, Cellerino A. Effects of dietary restriction on mortality and age-related phenotypes in the short-lived fish Nothobranchius furzeri. Aging Cell 2009;8(2):88–99. [53] Muller HJ. Artificial transmutation of the gene. Science 1927;66 (1699):84–7. [54] Sega GA. A review of the genetic effects of ethyl methanesulfonate. Mutat Res 1984;134(2–3):113–42. [55] Valenzano DR, Kirschner J, Kamber RA, Zhang E, Weber D, Cellerino A, et al. Mapping loci associated with tail color and sex determination in the short-lived fish Nothobranchius furzeri. Genetics 2009;183(4):1385–95. [56] Kawakami K, Shima A, Kawakami N. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc Natl Acad Sci U S A 2000;97(21):11403–08. [57] Suster ML, Kikuta H, Urasaki A, Asakawa K, Kawakami K. Transgenesis in zebrafish with the tol2 transposon system. Methods Mol Biol 2009;561:41–63.
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[58] Sumiyama K, Kawakami K, Yagita K. A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection. Genomics 2010;95(5):306–11. [59] Chapman JR, Taylor MR, Boulton SJ. Playing the end game: DNA doublestrand break repair pathway choice. Mol Cell 2012;47(4):497–510. [60] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337(6096):816–21. [61] Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science 2010;327(5962):167–70. [62] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339 (6121):819–23. [63] Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 2013;31(3):227–9. [64] Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013;153(4):910–8. [65] Harel I, Valenzano DR, Brunet A. Efficient genome engineering approaches for the short-lived African turquoise killifish. Nat Protoc 2016;11(10):2010–28. [66] Clark KJ, Urban MD, Skuster KJ, Ekker SC. Transgenic zebrafish using transposable elements. Methods Cell Biol 2011;104:137–49. [67] Dolfi L, Ripa R, Cellerino A. Transition to annual life history coincides with reduction in cell cycle speed during early cleavage in three independent clades of annual killifish. Evodevo 2014;5:32. [68] 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(7589):184–9. [69] Martin-Montalvo A, Mercken EM, Mitchell SJ, Palacios HH, Mote PL, Scheibye-Knudsen M, et al. Metformin improves healthspan and lifespan in mice. Nat Commun 2013;4:2192. [70] 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(8):1246–56. [71] Philippe C, Gregoir AF, Janssens L, Pinceel T, De Boeck G, Brendonck L. Acute and chronic sensitivity to copper of a promising ecotoxicological model species, the annual killifish Nothobranchius furzeri. Ecotoxicol Environ Saf 2017;144:26–35. [72] Philippe C, Hautekiet P, Gregoir AF, Thore ESJ, Pinceel T, Stoks R, et al. Combined effects of cadmium exposure and temperature on the annual killifish (Nothobranchius furzeri). Environ Toxicol Chem 2018. [73] Caracciolo B, Xu W, Collins S, Fratiglioni L. Cognitive decline, dietary factors and gut-brain interactions. Mech Ageing Dev 2014;136–137: 59–69. [74] Smith P, Willemsen D, Popkes M, Metge F, Gandiwa E, Reichard M, et al. Regulation of life span by the gut microbiota in the short-lived African turquoise killifish. Elife 2017;6. [75] Polacik M, Blazek R, Reichard M. Laboratory breeding of the short-lived annual killifish Nothobranchius furzeri. Nat Protoc 2016;11(8):1396–413.
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. 27, 2018
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
Models for Aging Research
The African turquoise killifish Nothobranchius furzeri as a model for aging research Hanna Reutera,1, Johannes Kruga,1, Peter Singera,1, Christoph Englerta,b,* a
Molecular Genetics Lab, Leibniz Institute on Aging–Fritz Lipmann Institute (FLI), Beutenbergstr. 11, 07745 Jena, Germany Institute of Biochemistry and Biophysics, Friedrich-Schiller-University Jena, Hans-Knöll-Str. 2, 07745 Jena, Germany
b
Nothobranchius furzeri is an African killifish with an exceptionally short median life span of 3 to 7 months. Despite its short life span N. furzeri displays signs of
Section editor: Dr. Hildegard Mack – Institute for Biomedical Aging Research, University of Innsbruck, Austria.
aging that are highly reminiscent of mammalian aging. In 2015, reference sequences for the N. furzeri genome have been published. In addition, transgenesis and genomic engineering of killifish using CRISPR/Cas9 have been recently established. It has also been shown that N. furzeri is amenable to pharmacologic intervention. In this review we will discuss what makes N. furzeri a valuable model for research on aging and whether it could also be used for drug discovery.
Introduction With the rise in life expectancy research on aging and agingassociated diseases is drawing increasing attention. In fact, while many disciplines within the biomedical sciences have sporadically considered aspects of aging, aging research has meanwhile become a discipline of its own. This becomes evident by the foundation of a number of institutes that are dedicated exclusively to research on aging. This process has started with the Buck Institute for Research on Aging in Novato, California and was followed by a number of insti*Corresponding author: C. Englert (Christoph.englert@leibniz-fli.de) 1 These authors contributed equally to the manuscript.
tutes in the US, Europe and Asia. Traditionally, the main model organisms in research on aging are yeast, Caenorhabditis elegans, Drosophila and the mouse. Recently, these wellestablished models have been joined by exceptionally longas well as short-lived vertebrates as new aging models, namely the naked mole rat [1] and the African killifish Nothobranchius furzeri [2,3]. The latter lives in seasonal ponds in southeast Africa and is the shortest-lived vertebrate that can be kept in captivity [3]. Depending on the strain N. furzeri has a median life span between 3 and 7 months and displays rapid growth and early sexual maturation [4,5]. What makes N. furzeri particularly appealing is established transgenesis [6–8] and genome editing [9] and the recent publication of a reference sequence for its genome [10,11]. In this review we discuss why N. furzeri is a valuable model for research and consider how killifish might be used for discovery and characterization of drugs that decelerate the aging process.
N. furzeri: life cycle and life span With less than one year of maximum life span, N. furzeri is an ideal model for research on aging. The short life span is
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considered to be an adaptation to its ephemeral natural habitat in South-Eastern Africa with alternating rainy and dry seasons. The fertilized killifish eggs and developing embryos survive extended dry seasons in an arrested state, called diapause that can occur at three different phases of embryonic development. To date, we still know very little about the mechanisms that trigger onset and duration of each diapause, however, low temperature and low oxygen seem to favor the onset of diapause, at least in N. guentheri [12]. When the rainy season begins, the embryos hatch and the larvae rapidly grow to sexual maturity (Fig. 1A,C), which can be reached as early as 14 days after hatching [5]. Currently, several N. furzeri strains that differ in origin and life span are used for biomedical research. The GRZ strain was originally isolated in 1969 in a semi-arid habitat in Zimbabwe, has a maximum life span of 4–6 months and is highly inbred [3,13,14]. Strains from more humid regions in Mozambique were established only several years ago, are genetically heterogeneous and have a longer maximum life span of 40–60 weeks [15,16]. Thus, the life span of N. furzeri is exceptionally short compared to other fish species that are used to model human diseases and aging like zebrafish or medaka with median life spans of 60 and 22 months, respectively [17,18]. In a very recent field study it was shown that the fast growth and rapid sexual maturation of N. furzeri that is observed in captivity is probably an underestimate of the timing of the natural processes. Natural populations that were surveyed by measuring otolith growth and gonad histology grew within two weeks from 5 to 54 mm and reached sexual maturity after 14 days [5]. Most likely, this is the fastest rate of sexual maturation in a vertebrate. The availability of natural populations and laboratory strains that differ largely in life span has enabled a quantitative trait locus (QTL) analysis regarding loci that determine life span. To this end a short- and a long-lived strain were crossed, life span was recorded and a QTL analysis was performed [19]. By combining genotype and phenotype information, four life span QTLs which taken together explain 27% of the total life span variance in the F2 generation were identified. The genes that are contained within the respective genomic regions can be considered as candidate genes for the determination of vertebrate life span.
dase and lipofuscin as well as the cell cycle inhibitors p21 and p16 have been shown to accumulate during N. furzeri aging [24,25]. Again, these markers are found in all other vertebrate aging models and are present in humans [26]. On an organismic level, aging in N. furzeri manifests itself by reduced coloration in males, malformations of the spine and face, and weight loss (Fig. 1B). These phenotypic changes are paralleled by changes in behavior, e.g. locomotor activity, open-field exploration as well as learning and memory function [24,27,28]. In addition, an age-dependent decay in adult neurogenesis has been described for N. furzeri [29]. This suggests aging-associated exhaustion of neuronal stem cells, which is another important hallmark of aging. Loss of regenerative capacity has also been observe with regard to the N. furzeri caudal fin [16]. Other hallmarks of aging have recently been proposed, including loss of proteostasis, epigenetic alterations and altered intercellular communication [26]. While direct evidence for some of those has not yet been obtained in N. furzeri, there is no reason to believe that they are absent or cannot be studied in killifish. Perhaps surprisingly, N. furzeri displays a high incidence of tumors, particularly in the liver and the kidney [30]. It should be noted, however, that in fish the kidney is the main hematopoietic organ. Therefore, it is likely that tumors in the kidney are leukemias rather than tumors of the kidney itself. It is still unexpected to find a high cancer rate in such a short-lived organism and suggests that killifish might also serve as models for tumorigenesis. Finally, the short life span of N. furzeri makes longitudinal studies feasible. In a first study of such kind gene expression during early life was correlated with individual life span [31]. One of the key results of this study was that expression levels of several gene sets, including oxidative phosphorylation, showed lower expression levels early in the life of long-lived individuals. This raised the hypothesis that expression of genes encoding components of the respiratory chain might be negatively correlated with individual life span. It was indeed shown in a subsequent experiment that treatment of N. furzeri with a low dose of rotenone, an inhibitor of complex I reverted gene expression signatures to those of younger age and led to extension of life span by 15%.
Hallmarks of aging in N. furzeri
Degeneration and regeneration
Despite its short life span N. furzeri shows many hallmarks that are typical signs of mammalian aging at the molecular, cellular, organ as well as organismic level including behavior. Telomere shortening is one of those hallmarks that have been reported to accompany aging in N. furzeri [20]. This is particularly interesting as in terms of length killifish telomeres are much more similar to human telomeres than the much longer mouse telomeres [21,22]. Also, mitochondrial dysfunction has been reported to occur in killifish upon aging [23]. In terms of molecular senescence markers, b-galactosi-
Repair processes are limited to a few organ systems in humans. Importantly, processes linked to regeneration such as wound healing often decline with age. The rate of reepithelialization after wounding is strongly affected in old humans [32] and children display better regrowth of lost fingertips than adults [33]. Regenerative therapies are a promising approach to reverse aging damage and restore functionality in the elderly. Degeneration and respective regeneration processes are complex, involving the interplay of many different cell types and factors that are difficult to fully model in
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Drug Discovery Today: Disease Models | Models for Aging Research
(a)
time
GRZ-D
growth
hatching (3 days)
adult (11 weeks)
(b)
time
MZCS-08/122
aging
young (3 weeks)
adult (19 weeks)
old (50 weeks)
(c)
<1 year
egg hatchling young adult old
pigmentation loss spinal curvature reduced activity learning deficit weight loss decreased fecundity liver neoplasia
telomere shortening mitochondrial dysfunction increase in aging markers (lipofuscin, senescence) oxidative stress (heart) neurodegeneration decreased regeneration (fin) Drug Discovery Today: Disease Models
Fig. 1. Rapid growth and aging in N. furzeri. (A) Rapid growth of N. furzeri GRZ (median life span of 3–7 months). For fish breeding, the dry/wet season conditions are mimicked in the lab whereby the fertilized N. furzeri eggs are collected on peat moss plates. While the hatchling is only a few millimeters of size, the adult fish reach a size of several centimeters. The colorful males are bigger in size than the grey female fish. Scale bar is 1 cm. (B) Representative pictures of young, adult and old male fish illustrate phenotypical changes with age (MZSC-08/122 strain that has a mean life span of about 11 months). Signs of aging on a phenotypical as well as molecular level are listed. Scale bar is 1 cm. (C) Schematic illustration of the rapid increase in body size with age.
vitro. The need of animal models to understand such processes is therefore of great importance. In contrast to humans, fish have been reported to have extraordinary regeneration capacities. The most prominent example is zebrafish that is
known to repair complex organs such as the brain, spinal cord, kidney, heart and appendages after severe injury (reviewed in Ref. [34]). Regeneration capacities of the caudal fin as well as the kidney have been reported for N. furzeri www.drugdiscoverytoday.com
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[16,35]. Functionality of the killifish kidney is restored faster after damage than in zebrafish: reabsorption of a fluorescently labeled sugar is almost back to normal already 8 days post injury, while kidney function recovery is only observed 2–3 weeks post injury in D. rerio [35–37]. Apoptotic levels peaked in renal tubules 2 days post injury in N. furzeri, which was accompanied by an increase of proliferating cells in tubules. Newly developing nephrons (neonephrogenesis) could already be observed 4 days post damage. This study suggests that tubular regeneration allows fast functional recovery in N. furzeri and is followed by neonephrogenesis [35]. Whereas repair of the damaged kidney by surviving tubular epithelial cells is common to mammals as well [38], neonephrogenesis seems to be distinctive to teleost fish. N. furzeri therefore offers the great opportunity to study molecular processes underlying the formation of new tubules after kidney damage, especially in an aging context. Caudal fins of young adult N. furzeri are nearly completely regenerated within 27 days after amputation, which is similar to the restoration of fin shape and size after 3 weeks in zebrafish [39]. Interestingly, an age-dependent decline in fin regenerative capacity has been reported in N. furzeri where fish with a mean age of 54 weeks (long-lived MZM-0703 strain) only regenerated 46% of their original fin size [16]. However, the observation that young fish regenerate faster than old fish has already been noted in Pierre M.A. Broussonet’s first description of fin regeneration in the 18th century when he experimented on goldfish [40]. For zebrafish, there are conflicting reports regarding agedependency. While one study reported life-long regeneration capacities for the zebrafish fin [41], other studies suggest that old animals show impaired fin regeneration [42,43]. A general drawback of the zebrafish compared to the killifish studies is a less careful definition of age groups. This might be a possible reason for opposing results. Zebrafish over 24 months of age are often considered as ‘old fish’ although they can live up to 5– 6 years. The short life span and the clear results concerning agedependent decline in regeneration capacities make N. furzeri the more powerful model system to study age-dependent regeneration, especially since animals from the same clutch can be more easily studied over time. For the killifish fin, molecular differences concerning age-dependency have been identified, whereas the zebrafish studies that reported agedifferences mainly compared macroscopic differences. Early key events during regeneration in old and young animals were identified to be different in killifish fins post amputation. Old fish showed a delay in bone formation, proliferation levels were higher in young fish fins while apoptotic cell numbers were increased in the regenerating fin of old fish [16]. Expression analysis of genes involved in fin regeneration showed an increased expression post amputation in all age groups studied, but older fish show a delay in peak expression of several genes (shh, fgf20a, fgfr1, wnt10a, raldh2, lef1 and msxb) [16]. 18
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The study that reported life-long regeneration capacities for the zebrafish fin also suggests the same for the zebrafish heart [41]. Since heart failure is an important health issue in elderly humans, it would be interesting to have an agedependent regeneration model for the heart as well. The neonatal mouse heart possesses a transient regenerative potential but the adult mouse heart lacks such regenerative capacities [44]. The human heart fails to regenerate after injury but cardiomyocyte renewal has been observed in young humans, although at low rate, while it is almost neglectable in adults [45,46]. With regard to heart failure and replacement of damaged heart muscle, this observation of cardiomyocyte proliferation in young humans is promising for the development of regenerative strategies in elderly. Whether the killifish heart is an appropriate system to study the aging heart remains elusive. The killifish heart is still poorly studied. Post-mortem analyses indicated an aging phenotype such as hypertrophy and heart dilation [30]. A study comparing old and young killifish hearts reported an increase of oxidative stress and changes in cardiac miRNA expression with age in killifish hearts [47]. It might be questionable whether the killifish heart responds similarly to damage as the zebrafish heart since the closer relative medaka shows impaired heart regeneration [48,49]. The nervous system is another important aspect concerning degeneration and regeneration processes. Neurodegeneration such as neurofibrillary degeneration and accumulation of the age-related pigment lipofuscin in the brain have been reported for N. furzeri [27,50]. This observed neurodegeneration is in line with decay in cognitive function assessed by open field exploration and learning performance in a shuttle box [27,50,51]. Interestingly, dietary restriction was able to reduce neurodegeneration and helped to improve learning performance [52]. This observation indicates that N. furzeri is a promising model to find tools to stop degeneration and restore regeneration capacities in the elderly. More organ systems in N. furzeri need to be investigated for their regeneration capacity in future to expand models for human degenerative diseases. In order to understand how regenerative strategies can potentially be applied in humans to fight aging damage, it is of special interest to study regeneration processes in an age-related context. Only by using such a model system that allows us to combine regeneration and age research, we will be able to elucidate which cellular and molecular processes underlie regeneration in general and the observed decline in regenerative capacities with age.
Genome engineering in N. furzeri Gene inactivation is state-of-the-art to study gene function in vivo. The classical forward genetics approach is based on the random generation of mutations followed by the subsequent analysis identifying the genetic alteration causing the observed phenotype. Those random mutations were induced by
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genome sequence can be found in the N. furzeri Genome Browser (NFINgb) as well as the African Turquoise Killifish Genome Browser. Further access to sequence and transcriptome data is possible via BLAST and the N. furzeri transcriptome browser (NFINtb) allowing for comparisons with other species. All target-specific genome-editing systems are based on the induction of a DNA double-strand break in the locus of interest stimulating the DNA damage response pathways. Those double strand breaks can either be repaired via the error-prone non-homologous end joining (NHEJ) or the highfidelity homology-directed repair (HDR) mechanism [59]. The introduction of the CRISPR/Cas9 technology in 2012 started to revolutionize the field of sequence-specific genome-editing, since it is cheaper and easier applicable than the earlier employed zinc-finger nucleases (ZNFs) and transcription activator-like effector nucleases (TALENs) [60]. Naturally, the CRISPR/Cas system is the adaptive immune system in microbes, which recognizes and degrades foreign nucleic acids from viruses and bacteriophages [61]. Since its identification as a tool for genomic engineering, the CRIPSR/Cas technology has been widely applied in a broad range of model organisms starting with human and mouse cells [62], Danio rerio [63] and Mus musculus [64]. To achieve successful genome-engineering, sequence-specific single guide (sg)RNAs are guiding the Cas9 endonuclease to the sequence of interest, where a DSB is induced. In 2015, the first CRISPR/Casinduced knockouts were described in N. furzeri [9] (Fig. 2). Since N. furzeri has been established primarily as a model organism for aging research, the targeted genes were all linked to aging (e.g. igf1r, tert) in order to study the aging
the exposure to mutagens such as X-rays [53] or chemicals like ethyl methanesulfonate [54]. The nowadays commonly used reverse genetics techniques are used to firstly induce mutations in the gene of interest and then study its impact on the organism. However, this approach requires two main prerequisites: a sequence-specific genome editing tool as well as an assembled and annotated genome. With the help of the first genetic linkage map based on microsatellites [55] and a partial sequence of the N. furzeri genome obtained by Sanger sequencing [14] the first genetic modifications were possible. In 2011, a first protocol for transposon-mediated transgenesis in N. furzeri was published [8] (Fig. 2). This transgenesis approach is based on the Tol2 transposon system, which originates from Oryzias latipes (Medaka) [56], and has previously also been successfully applied in other model organisms such as zebrafish [57] and mouse [58]. With this system a construct flanked with Tol2 recognition sites can be inserted randomly into the genome. Due to the thick chorion surrounding the N. furzeri egg, microinjection protocols had to be adapted in order to get access to the cell for the injection [7,8]. Based on those protocols the first transgenic N. furzeri lines expressing GFP under the control of the Cska or Hsp70 promoter were generated, allowing ubiquitous as well as inducible expression of transgenes such as a heat-shock induced reversible expression of GFP [6,8] (Fig. 2). Tools for sequence-specific gene modifications have been established during the last years and were already frequently applied in various model organisms. Together with the publication of the N. furzeri (1.24 Gb) genome in 2015 [10,11], this was a milestone for sequence-specific genome engineering in N. furzeri (Fig. 2). The N. furzeri
Nothobranchius furzeri as model for aging research
First insights into the N. furzeri genome
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Husbandry protocol
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Regeneration of the fin and kidney
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Fig. 2. Hallmarks in Nothobranchius furzeri research. During the last 15 years, N. furzeri has been established as a model organism for aging research. The scheme summarizes important hallmarks in the research on the short-lived African killifish. Pictures adapted from Ref. [7]. www.drugdiscoverytoday.com
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process and age-associated diseases. As an example a telomerase-deficient tert knockout line was generated to analyze telomere attrition during aging. Besides the short life span N. furzeri has telomeres of a similar size range as humans, in contrast to mice that have to be bred for several generations until the occurrence of telomerase-related phenotypes [9]. Besides the knockout of several genes, knock-in experiments using the CRISPR/Cas9 system in the short-lived killifish were also performed [9,65]. Instead of the error-prone NHEJ, the precise homology-directed repair (HDR) mechanism is needed to repair the Cas9-induced double-strand break. For this purpose a single-strand DNA template needs to be provided that can then be inserted at the desired region of interest. This HDR-based engineering can be used to insert exogenous DNA sequences or precisely alter genomic sequences at specific target sites. By this, specific human disease-causing mutations of tert have been introduced in N. furzeri [9]. Moreover, this technique could be applied to insert for example loxPsites or reporter genes into specific loci to further expand the experimental applications of N. furzeri. The short life span of N. furzeri comes along with a rapid growth and an early sexual maturity after only two to three weeks in the short-lived strain [4,5]. This allows a rapid generation of stable genetically altered N. furzeri lines within two to three months [9,65]. In contrast, for the generation of a stable D. rerio line, a time span of 2.5–4 months is already needed to reach sexual maturity in F0 fish [66]. Despite its rapid growth, the embryonic development of N. furzeri is very slow [67]. The one-cell stage in N. furzeri, used for microinjections, can last up to a few hours, greatly increasing the chances of an early induction of the genomic modification and thereby reducing mosaicism. Both, the availability of an annotated genome as well as established genomic modification protocols make N. furzeri a suitable model organism for various biological research fields. Genomic alterations in N. furzeri are currently mainly focusing on aging and aging-related disease. Additionally, specific modifications of receptors, genes involved in regulatory pathways and others can be used to study impacts and effects of specific drugs.
Influences on life span by environmental conditions and drugs In recent years two different strategies have been employed to delay aging and the onset of aging-associated diseases, namely genetic and pharmacological approaches [68,69]. Of late, senolytic drugs that can selectively eliminate senescent cells have come into focus [70]. In this regard, the question arises whether N. furzeri might be an appropriate model for drug testing. The drug resveratrol, which has been shown to retard aging in a number of aging models like yeast, worm and fly, has indeed been shown to prolong life span and delay the onset of aging markers in N. furzeri [50]. Another intervention 20
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that has been shown to increase life span in N. furzeri was reducing the water temperature [28]. Also, the effect of dietary restriction (DR) on aging and longevity has been tested in two different lines, namely an inbred and a longer-lived wildderived line of N. furzeri [52]. While DR prevented the occurrence of the age marker lipofuscin in the brain and improved performance in a learning test there were differential effects regarding life span. Whereas in the inbred strain overall agerelated risk of mortality was reduced, in the wild-derived strain DR even induced early mortality. In both lines DR extended maximum life span, albeit to different degrees. In terms of drugs, the only other substance that had been administered to N. furzeri to date is rotenone, an inhibitor of mitochondrial respiratory chain complex I [31]. Rotenone extended life span by 15% and reversed aging-associated gene expression profiles in different organs toward younger age. This effect is most likely a hormetic response, as a higher dose of rotenone in N. furzeri was life-shortening [31]. Of note, killifish has recently also been used for ecotoxicological testing regarding for example the effects of cadmium and copper [71,72]. Although reports on pharmacological screens are still scarce, the application of resveratrol and rotenone on N. furzeri show that this species is well-suited for the identification of pharmacological modifiers of aging and regeneration. Because of its territorial behavior, particularly male N. furzeri animals are often kept in single housing tanks. Also, most killifish facilities operate with a system in which multiple tanks are connected. This limits the possibility to perform large-scale pharmacological screens. However, the accessibility of several aging markers enables small- and mid-scale pharmacological screens with the goal to identify drugs, which delay aging and the onset of aging-related diseases. The killifish model is therefore best suited as a secondary screening model in which a certain number of candidate drugs that have been obtained by a primary screen (e.g. in cell culture) shall be validated in a vertebrate model.
Outlook A promising approach to find interventions for aging in humans and to prolong health span is to combine the described genomic editing, regeneration research and drug screening approaches described in this review. On this route, N. furzeri with several natural strains of different life span, the possibility to edit its genome, the availability of its genome sequence and the possibility to perform genetic and pharmacological screens for genes and substances affecting aging and longevity is emerging as a very promising model. Recently, the microbiota, particularly the gut microbiota has been shown to influence multiple processes in vertebrates including aging [73]. Also in this regard, N. furzeri turns out to be a very useful model [74]. It was shown that transferring the gut microbiota from young to middle-aged individuals prevented behavioral decline and extended life span.
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Finally, to further increase the acceptance of N. furzeri as a model for different research fields, it is necessary to establish general housing and breeding conditions for this species as has been suggested earlier [75]. Additionally, current efforts to develop a standardized diet aim to minimize the risk of contamination caused by the widely used live food. The Nothobranchius Symposium, held every two years, serves as a platform for the exchange of knowledge and improvements concerning research and husbandry.
Acknowledgements We want to thank all members of the Nothobranchius project at FLI for their contributions and particularly thank the fish facility for excellent fish care over the years. The authors also acknowledge continuous support from the Leibniz Association, the German Federal Ministry of Education and Research (BMBF), and the German Research Foundation (DFG).
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