Sci. Aging Knowl. Environ., 28 August 2002
Vol. 2002, Issue 34, p. re4
[DOI: 10.1126/sageke.2002.34.re4]


Subfield History: Caenorhabditis elegans as a System for Analysis of the Genetics of Aging

Thomas E. Johnson

The author is in the Institute for Behavioral Genetics, University of Colorado at Boulder, Boulder, CO 80309-0447, USA. E-mail: johnsont{at}Colorado.EDU;2002/34/re4

Key Words: Caenorhabditis elegans • genetics • age • longevity genes • aging genes

Abstract: This article reviews key events in the genetic analysis of aging in the worm. The events are presented in the form of a timeline and include landmark papers, key meetings, and the development of important funding agencies. I also speculate on events that might appear in this timeline if this review were written in the distant future.

Introduction Back to Top

Research on aging has a long history, dating almost from the beginning of recorded time. The Epic of Gilgamesh of ancient Babylon, written in about 2000 B.C. in cuneiform (Fig. 1), tells of the hero's quest for immortality. This innate desire for extended life has been both a boon and bane to research in the field of aging. To this day, hyperbole surrounding research results and the potential to intervene in and extend life-span plague the field of aging (see Olshansky Perspective and "Gero-Tech Sprouts, But Will It Bloom?").

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Fig. 1. The Epic of Gilgamesh of ancient Babylon, written in about 2000 B.C. in cuneiform. © Copyright The British Museum.

Research in the field of aging has been revolutionized by the implementation of a genetic approach to the analysis of life-span specification. The past two decades have seen the identification of more than 50 genes that lengthen life-span. Such discoveries have been made in a variety of model species, but studies in the nematode Caenorhabditis elegans have been especially fruitful. What follows is an account of the most significant findings about the specification of life-span in C. elegans. This review is intended to provide background on material that is largely unavailable in more recent reviews on the genetic specification of aging. For a more personal retrospective about the study of aging in C. elegans, the reader is referred to my recently published article. For a lively commentary on the state of senescence research, I refer the reader to a recent article by George M. Martin (SAGE KE's Editor-in-Chief).

The 1970's Back to Top

Event (1974): The National Institute on Aging (NIA), one of the National Institutes of Health (NIH), is founded. Much of the financial support for research on the genetics of C. elegans (both as it applies to aging and to other aspects of worm biology) has been through the NIA. The founding of the NIA meant that for the first time, aging could be viewed as a process that occurs independently of the pathological consequences of aging, as revealed by studies of human diseases. To appreciate how prescient this view was, consider that even now, in 2002, the existence of an aging process independent of disease remains controversial (see Winker and Olshansky).

Here is an exerpt from the History and Mission of the NIA: "The NIA, one of the 25 institutes and centers of the NIH, leads a broad scientific effort to understand the nature of aging and to extend the healthy, active years of life. In 1974, Congress granted authority to form the NIA to provide leadership in aging research, training, health information dissemination, and other programs relevant to aging and older people. Subsequent amendments to this legislation designated the NIA as the primary federal agency on Alzheimer's disease research."

Robert N. Butler was the Director-Designate of the NIA when it was first created. To read his reminiscings about the early days of the NIA, click here.

Funding (1975): The NIA issues a Program Announcement (PA), Genetics and Comparative Aging, specifically mentioning C. elegans as a target for funding by the NIA. This PA was championed by Don Murphy, who had the early vision of the potential value of C. elegans in research on aging. PAs are statements of intent to fund certain areas of research and have the goal of soliciting research proposals in the targeted areas.

Publication: Michael Klass and David Hirsh, Non-ageing developmental variant of Caenorhabditis elegans. Nature 260, 523-525 (1976).

The authors demonstrated that dauer larvae can be maintained as dauers for at least 2 months and still retain a normal life expectancy and fertility when returned to normal growth conditions. Thus, dauers are a "time out" from the normal aging process.

Dauers or dauer larvae are an alternative developmental form to the normal third-stage larva. They are formed when conditions deteriorate (when there is a lack of food, high temperature, etc.) or there is significant crowding. Dauers are very resistant to environmental stress. For information about dauers, see Don Riddle's and Gary Ruvkun's Web sites.

Publication: Michael Klass, Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life-span. Mech. Ageing Dev. 6, 413-429 (1977).

This classic study paved the way for subsequent work that identified genetic variants and mutants that slow the rate of aging. Klass described key environmental factors that influence life-span in C. elegans. Life-span was shown to be dependent on temperature [between 16° and 25.5°C, Q10 (the ratio of the metric at two temperatures separated by 10°C) = 2.7] and food concentration (108 bacteria/ml is the optimal concentration for maximum longevity, but there is a severe reduction in fertility at these concentrations). Fertility, fecundity, and oocyte production were similarly assessed under various environmental conditions (20°C is optimal). The nematode was also shown to become less sensitive to ultraviolet radiation after it had completed development. Klass also tested numerous theories of aging that had been proposed at the time, including evolutionary trade-offs between reproduction and longevity (this was inconsistent with his data); increase of life-span by dietary restriction (this was confirmed by his data); parental age effects on life-span (progeny from older parents lived for a slightly shorter amount of time than did progeny from younger parents); and heritability of life-span (long-lived parents had slightly longer-lived progeny; no mutagen was employed). He also quantitated the production of lipofuscin-like material with increasing age. Lipofuscin is a pigmented product that accumulates as the worm ages; these aggregated polymers are derived from the oxidation products of proteins and lipids.

Conference: Genetic Effects on Aging, I (1978). This is the first of three meetings initiated by the Jackson Labs that focused on the genetic effects on aging. Two more meetings have followed in this series, in 1988 and 2000. David Harrison, who is still at the Jackson Labs, was the driving force. The meeting was held in Bar Harbor, Maine and was sponsored by The March of Dimes. There was quite a bit of time for discussion at this meeting, and much of it is summarized in the book published from the proceedings [D. Bergsma, D. E. Harrison, Genetic Effect on Aging (Liss, New York, 1978)]. Many of the contributions are worth reading today.

George Martin delivered his classic paper on segmental progeroid syndromes in humans [G. M. Martin, Genetic syndromes in man with potential relevance to the pathobiology of aging. Birth Defects Orig. Artic. Ser. 14, 5-39 (1978)]. A noteable quote from his chapter (p. 35) is, "I have emphasized that none of these [progeroid] syndromes can in any way be regarded as 'acceleration phenocopies' of the entire spectrum of what is normally found in senescence."

Conference: Gordon Conference on the Biology of Aging: Genetic and Evolutionary Aspects of Aging (1979). This was the first Gordon Conference on the Biology of Aging that prominently featured genetics (Fig. 2). It was held in Santa Barbara, CA, and George Martin was the chair of the meeting. The meeting focused on comparative evolutionary approaches and prominently featured several discussions of genetic mutations that affect longevity and other aspects of aging in C. elegans. Martin had this to say about the meeting: "I remember that there was a special lecture by Steve Gould and that this was the first Gordon Conference on aging attended by Tom Kirkwood. It was also the first one with a theme on 'Model Systems for Aging Research Amenable to Genetic Analysis.' There were also sessions on C. elegans, Drosophila melanogaster, fungi (Neurospora), mice, and man."

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Fig. 2. Photo of the participants in the 1979 Gordon Research Conference on the Biology of Aging. To see photo key and agenda, click here.

On a personal note, this was the first meeting on aging that I attended, as I had just made the decision to switch my research focus from development to aging.

1980-1984 Back to Top

Book: Bert M. Zuckerman, Ed., Vol. 1: Behavioral and Developmental Models and Vol. 2: Nematodes as Biological Markers: Aging and Other Model Systems (Academic Press, New York, 1980).

This book contains numerous chapters that review a variety of important aspects of normal nematode development, but especially the biological processes involved in aging. It served as a key resource in the early days of C. elegans genetics, before the publication of the first book on C. elegans in the Cold Spring Harbor series.

Publication: Tom Johnson and Bill Wood, Genetic analysis of life-span in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 79, 6603-6607 (1982).

Johnson and Wood published the first clear evidence that genes control aging and longevity. To accomplish this, they applied classical quantitative genetic methodology to the analysis of longevity. They intercrossed two wild-type strains (N2 and Bergerac) and examined F1 and F2 progeny for longevity. They found a lack of heterosis (hybrid vigor) in the F1 progeny. This finding makes sense when considered in the context of the self-fertilizing mode of reproduction in C. elegans, which continually renders the genome homozygous, thus eliminating recessive deleterious alleles that probably underlie the hybrid vigor observed in outbreeding species. They also made recombinant inbred (RI) strains, which are stable inbred strains that each contain, on average, 50% of each parent's alleles. These RI strains have since been used by many other investigators to study quantitative traits that specify aging and aspects of the animal's life history and have been especially important in behavioral genetics. The major advantage of RI strains over other types of segregating populations, such as F2 populations, is their stable genotype, which allows comparisons between replicate experiments conducted in different labs at different times. This has led to the recommendation that they be used for further polygenic analyses.

Johnson and Wood used several independent methods to estimate the heritability (the fraction of the variation in life that can be ascribed to genetic effects) of life expectancy. They proposed that genes that prolong the life-span of wild-type animals must specify "primary rate-limiting processes that determine life-span." This work was the first paper on the genetics of aging in C. elegans that was published in a prestigious mainstream journal.

Publication: Michael R. Klass, A method for the isolation of longevity mutants in the nematode Caenorhabditis elegans and initial results. Mech. Ageing Dev. 22, 279-286 (1983).

Klass demonstrated for the first time in any metazoan species the existence of mutants with longer life expectancy. This was an extremely significant piece of work. Few mutant genes other than age-1 have been found using only extended longevity as a screen, because of the difficulty of using longevity as a phenotype. Klass screened for extended longevity in replicate cultures in 20-well microtiter plates. To keep his screening populations free of progeny, he used a temperature-sterile mutant in which sperm formation is blocked at 25°C. This method is still widely used to block fertility. An alternative method of blocking fertility, which uses fluorodeoxyuridine, is also widely used but has some reported drawbacks that remain unsolved.

These mutants were not examined genetically by Klass, and they displayed multiple phenotypes, which led many to be skeptical that these were single-gene mutants. (Klass himself concluded that there were no genes that specifically regulate life-span. His mutants, which were not outcrossed, showed uncoordinated behavior, which Klass interpreted to mean that they consumed less food than normal. So he concluded that they lived longer because they were calorically restricted.) The fact that different isolates of age-1 contained an unlinked unc-31 mutation argues that they were not independently derived, although they have been given separate allele numbers (hx542 and hx546). Subsequent studies of the Klass mutants showed that all of them were age-1.

There was considerable doubt that mutations in a single gene could prolong life. A quote from George Martin's 1978 essay summarizes this sentiment: ". . . it is na�ve to believe that a mutation at a single locus could be responsible for the determination of life-span and the various debilities of aging." [G. M. Martin, Genetic syndromes in man with potential relevance to the pathobiology of aging. Birth Defects Orig. Artic. Ser. 14, 5-39 (1978)]. However, even today, many scientists fail to acknowledge the implications of age-1 and other mutants for evolutionary biology and effective therapeutic intervention.

Book: David H. Mitchell, Thomas E. Johnson, Eds., The Invertebrate Models in Aging Research (CRC Press, Boca Raton, FL, 1984).

This book tabulated much of the relevant background information on aging in C. elegans in a useful format and helped to make it more accessible to a wide range of people who might be interested in doing research on the genetics of aging.

1985-1989 Back to Top

Publication: Thomas E. Johnson, Aging can be genetically dissected into component processes using long-lived lines of Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 84, 3777-3781 (1987).

I demonstrated that aging in C. elegans consists of several distinct processes. Using RI strains, I was able to demonstrate independent segregation of a number of polymorphisms that differentially specify life-span, rate of development, length of the animal's fertile period, levels of reproduction, and age-specific behaviors. These results allowed me to conclude that each of these processes is under differential genetic control. This study was a precursor to the use of longevity mutants in quantitative trait loci (QTLs) mapping (in a variety of organisms, including worms, flies, mice, and humans) and in the genetic dissection of life history. Several labs, including my own, have followed up on this approach to map QTLs for these various traits. This paper also proposes a biomarker that accurately predicts age at death: the rate of loss of movement with chronological age.

Publication: D. B. Friedman, T. E. Johnson, A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility. Genetics 118, 75-86 (1988).

In this paper, the first mutant gene that specifies longer length of life (age-1) was named and mapped to linkage group II. These mutants were identified by Klass in 1983 and sent to us when Michael left academic science for the business world. We formally demonstrated that the mutant phenotype segregated as a single gene and had a stable phenotype, and then went on to show that development was normal in these mutants. The demonstration that a long-life phenotype could result from a single mutation was very controversial (see the 1983 publication by Klass above), but showing that age-1 could be mapped and that it segregated as a single gene helped convince many of this surprising fact. A shortcoming of the paper is that the age mutants were not extensively outcrossed to remove other mutations. Thus, although the unlinked unc-31 mutation was removed, the fer-15 mutation, which was used to sterilize the worms, was not removed from any progeny, because it is also on linkage group II. This cosegregation led to some confusion and an incorrect interpretation that age-1 mutants have a fivefold reduction in fertility in addition to a 40% increase in longevity. This erroneous conclusion might have resulted in the acceptance of the paper, because it confirmed the widely held belief that longevity genes should be pleiotropic, with life-span prolongation being associated with reduced fertility. Note that even today, all pro-longevity mutants found in any species show secondary effects on other aspects of development, behavior, or life history, a finding that is consistent with the phenomenon of negative pleiotropy (see Williams Classic Paper).

With respect to the age-1 mutants, Walker et al., 2000 showed that age-1 mutants are outcompeted by wild-type worms in an environment where populations are periodically allowed to starve; however, these slight differences in fertility were not observed in competition experiments where ample amounts of food were consistently present. They also showed that age-1 and wild-type strains have very similar developmental profiles.

Publication: D. B. Friedman, T. E. Johnson, Three mutants that extend both mean and maximum life-span of the nematode, Caenorhabditis elegans, define the age-1 gene. J. Gerontol. 43, B102-9 (1988).

This second paper in the age-1 series was largely devoted to the results of multiple complementation tests among the various mutants identified by Michael Klass. These experiments showed that all mutants failed to complement for their extended longevity (Age) phenotype. These results were published separately because Genetics suggested that these complementation data be removed from the original paper. The journal editor felt that the data were insignificant, and Klass had never claimed independent origin. Later, it was shown that the age-1 reference allele (hx546) failed to complement daf-23 (see Morris et al., 1996 ). Nevertheless, different allele numbers were assigned to age-1 mutant alleles from the different Klass strains. Interestingly, the mutational events in the age-1(hx546) reference allele and all other alleles from the Klass and Johnson labs remain undetermined.

Conference: Genetic Effects on Aging, II (1989). The second meeting on the Genetic Effects on Aging was again held at the Jackson Labs and organized by David Harrison. This time, two talks describing nematode experiments were given. Chapters describing the presentations, by Johnson et al. and Russell and Seppa are found in the conference proceedings, and both focus on studies using age-1 mutants. At this time, the Johnson and Russell labs were the only nematode labs funded for the study of aging.

1990-1994 Back to Top

Publication: T. E. Johnson, Increased life-span of age-1 mutants in Caenorhabditis elegans and lower Gompertz rate of aging. Science 249, 4908-4912 (1990).

In this paper, I demonstrated that the age-1(hx546) allele slows the "rate of aging" and is recessive. This was done by following survival curves on populations of 200 worms for each of four genotypes, including wild type and the age-1 mutant. Specifically, the experiments were performed with four groups of 50 worms and by different people and were done blind in a few instances. These data were replicated several times as well. We looked at age-specific mortality and found that age-1 mutants slowed the exponential rate of increase in mortality rate (sometimes called the Gompertz rate, after the 19th-century actuary who first noticed this effect in human populations) that has become a hallmark for slowing the actual process of aging. Before this paper, the only known way of slowing aging was dietary restriction. A key concept was that loss of or reduced gene function slows the rate of aging; thus, the aging rate is actually increased by the normal AGE-1 protein.

Review: R. L. Rusting, Why do we age? Sci. Am. 267, 130-135, 138-141 (1992).

This article provided general recognition of and popularized the genetics of aging field. This field had been largely overlooked and relatively unrecognized by much of the scientific establishment.

Funding (1992): The NIA initiates the Longevity Assurance Genes grant program for the support of basic research on the genetic and molecular bases of longevity. One of the key goals of this program was to discover genes that function in the determination of life-span. The discovery of an explosion of new alleles leading to life extension has resulted from this method of support, which brought many new investigators into the field. This program has been continued into the 21st century. Here is a recent program announcement from 1998.

Publication: Cynthia Kenyon, Jean Chang, Erin Gensch, Adam Rudner, Ramon Tabtiang, A C. elegans mutant that lives twice as long as wild type. Nature 366, 404-405 (1993).

This was a breakthrough paper for several reasons. First, Kenyon and her colleagues demonstrated that daf-2, a well-studied mutant, is also long lived and that this increased longevity is suppressed by the daf-16 mutation. Second, this finding introduced the key concept that aging is specified, at least in part, by a known pathway and suggested that the longer life of daf-2 mutant worms, and probably of worms with other life-extending mutations (gerontogenes), might result from the ectopic expression of a dauer-specific phenotype. Suddenly, age-1 was not an anomaly. Kenyon had made her scientific reputation studying C. elegans development. Having someone with her reputation working on mechansims of aging added considerable validation to the field. Recall that at this time, research on aging was conducted by a small number of scientists and funded by only a few grants. age-1 was not known to be in the dauer pathway at this time and was not discussed in this paper.

Animations of a wild-type versus a daf-2 mutant worm (young and old) can be seen here (courtesy of Douglas Crawford in the Kenyon lab).

Publication: Pamela L. Larsen, Aging and resistance to oxidative damage in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 90, 8905-8909 (1993).

In this paper, Larsen investigated whether conditions known to extend life-span (the dauer larva state and the age-1 mutation) also rendered worms resistant to cellular damage thought to be related to aging. The age-1 mutant worms showed increased resistance to oxidative stress and elevations in the activities of superoxide dismutase (SOD) and catalase, two enzymes that protect cells against oxidative damage. These findings suggest that age-1 functions as a negative regulator of these enzymes and support the free radical theory of aging. This theory predicts that aging is a result of oxidative damage to tissues caused by free radicals (see "The Two Faces of Oxygen").

Publication: Jacques R. Vanfleteren, Oxidative stress and ageing in Caenorhabditis elegans. Biochem. J. 292, 605-608 (1993).

This paper demonstrated that the increased longevity of age-1 and daf-2 mutants is associated with increased resistance to oxidative stress. The author also showed that these strains have increased SOD and catalase activity. These findings and those of Larson discussed above support the stress-resistance model for increased longevity and were the first findings of molecular correlates of increased longevity to be published.

1995-1999 Back to Top

Publication: Gordon Lithgow, Tiffany White, Simon Melov, Tom Johnson, Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress. Proc. Natl. Acad. Sci. U.S.A. 92, 7540-7544 (1995).

Lithgow and colleagues extended the range of stress resistance phenotypes of age-1 to include resistance to high (lethal) temperatures by showing that age-1 is more resistant than the wild type. They also showed that daf-2 and spe-26 (another class of longevity mutants) are heat resistant, which is consistent with increased stress resistance serving as a biomarker of increased longevity. This study also produced convincing evidence for "hormesis," in which shorter exposure to lethal (35° C) temperatures resulted in life extension by ~15%.

Publication: Simon Melov, Gordon Lithgow, Doug Fischer, Pat Tedesco, Tom Johnson, Increased frequency of deletions in the mitochondrial genome with age of Caenorhabditis elegans. Nucleic Acids Res. 23, 1419-1425 (1995).

Melov and colleagues showed convincingly that the frequency of mitochondrial DNA deletions increases with chronological age in C. elegans. They also showed that age-1 mutant worms display fewer mitochondrial deletions at advanced ages than do wild-type worms.

Publication: Bernard Lakowski, Siegfried Hekimi, Determination of life-span in Caenorhabditis elegans by four clock genes. Science 272, 1010-1013 (1996).

Lakowski and Hekimi discovered a new class of four age mutants, which he called Clk (clock). These mutants (clk-1, clk -2, clk -3, and gro-1) slow development and increase variability in developmental timing, but also have a modest effect on adult longevity (on the order of 20%). These authors were the first to use double-mutant analysis of different gerontogene mutants and showed that clk-1;age-1 or gro-1;age-1 double mutants live much longer than either single-mutant parent. Their interpretation of this result is that clk genes function independently of mutants in the age-1 pathway to specify increased longevity. They also observed an anomalous effect: that the increase in life-span seen in the clk mutants is shortened but (they argued) not suppressed by daf-16, and they interpreted this to mean that daf-16 is part of a distinct pathway that does not involve the clk genes. They speculated that the clk class of mutants is regulatory in nature and might have an independent role in determining longevity. This class of mutants has turned out to be heterogeneous in its molecular actions and seems not to result in regulatory alterations.

Publication: J. Z. Morris, Heidi A. Tissenbaum, Gary Ruvkun, A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans. Nature 382, 536-539 (1996).

These authors describe the cloning of the daf-23 gene, which encodes a homolog of the mammalian phosphatidylinositol-3-OH kinase (PI3K) catalytic subunit. Many mutational events were found within the PI3K coding sequence. They also showed that daf-23 mutants, which are constitutive for dauer formation, failed to complement age-1 for longevity and its 27°C constitutive dauer formation defect. These findings, together with genetic mapping data, show that daf-23 and age-1 are the same gene, and Ruvkun and his colleagues suggested that the daf-23 mutants are nullomorphic, whereas the age-1 alleles are hypomorphic. However, none of the age-1 mutant alleles from the Klass, Johnson, or Lithgow labs were found to correspond to changes in the coding sequence, leaving the molecular etiology of the weaker phenotype still unknown. This finding, together with the cloning of daf-2 (Kimura et al., 1997), led to the widely publicized homology of the daf-2 pathway in worms with the insulin/insulin-like growth factor-1 (IGF-1) signaling pathways in mammals and catalyzed a large body of research to fill in the missing pieces of the pathway, such as akt-1/2, pdk-1, and Pten/daf-18 (Fig. 3). These genes were cloned and shown to be involved in the daf-2 pathway by several labs using a variety of strategies, including knocking out of the candidate genes.

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Fig. 3. The DAF-2 pathway in C. elegans. The components of the DAF-2 pathway are shown. When growth conditions are favorable (Left panel), insulin-like ligands bind to DAF-2, an insulin/IGF-1-like receptor. Activated DAF-2 then phosphorylates AGE-1, a phophatidylinositol-3 (IP3) kinase, which in turn phophorylates IP2 to form IP3. DAF-18, an AGE-1 agonist, catalyzes the reverse reaction. IP3 and AGE-1 together catalyze the phosphorylation of PDK-1. Activated PDK-1 and IP3 then phosphorylate AKT-1 and AKT-2, which in turn phosphorylate DAF-16. Phosphorylated DAF-16 binds to a 14-3-3 protein and is held in the cytoplasm, unable to stimulate the transcription of target genes. Under stressful conditions (Right panel), the DAF-2 pathway is not activated, and DAF-16 remains phosphorylated and therefore cannot bind the 14-3-3 protein. DAF-16 is now free to enter the nucleus and stimulate the transcription of target genes. Following are the years in which the genes that encode the various gene products in the insulin/IGF-1 pathway were shown to have effects on longevity. The seminal papers are hyperlinked. AGE-1, 1983; DAF-2, 1993; DAF-16, 1993; AKT-1/2, 1998 (This paper shows that AKT-1/2 is necessary for insulin receptor-like signaling in C. elegans and functions primarily to antagonize DAF-16. But the paper does not show that AKT-1/2 is involved directly in the determination of longevity.); PDK-1, 1999; DAF-18, 1998; 14-3-3, 2001 (This paper shows that 14-3-3 is involved in the insulin/IGF-1 pathway but does not show that 14-3-3 is involved in the determination of longevity.)

Conference: Gordon Conference on the Biology of Aging (1997) (T. E. Johnson, organizer). This was the first meeting in the Gordon Conference series to focus exclusively on the genetics of organismic longevity (Fig. 4). Every major lab in the world was asked to present, and almost all were represented.

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Fig. 4. Photo of the participants in the 1997 Gordon Research Conference on the Biology of Aging. To see photo key and agenda, click here.

Publication: Koutarou D. Kimura, Heidi A.Tissenbaum, Yanxia Liu, Gary Ruvkun, daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277, 942-946 (1997).

This paper convincingly showed that daf-2 is homologous to the mammalian insulin and IGF-1 receptors. This paper is among the most cited papers in the body of literature on aging and has been responsible for much of the attention that research into the genetics of aging has been getting over the past several years. See also Morris et al., 1996, above.

Publication 1: S. Ogg, S. Paradis, S. Gottlieb, G. I. Patterson, L. Lee, H. A. Tissenbaum, G. Ruvkun, The Forkhead transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994-999 (1997).

Publication 2: K. Lin, J. B. Dorman, A. Rodan, C. Kenyon, daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319-1322 (1997).

These two papers demonstrated that daf-16 is a member of the Forkhead class of transcriptional regulatory proteins and is a close homolog of several mammalian transcription factors. These findings have been used to further our knowledge of mammalian gene regulation by the insulin/IGF-1 signaling pathway. These papers also revealed that mutations in daf-16 suppress the constitutive dauer formation and extend the longevity phenotypes of daf-2 and age-1, indicating that the products of these genes function in the same biological pathway. This paper completes the signal transduction pathway from DAF-2 (at the cell surface) to DAF-16 (in the nucleus) (Fig. 3). Activation of this pathway culminates in the expression and repression of genes regulated by DAF-16. Scientist are in the process of identifying these genes using genome-scale gene expression analysis.

Publication: Bernard Lakowski, Siegfried Hekimi, The genetics of caloric restriction in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 95, 13091-13096 (1998).

The authors revealed a new class of Age mutants by showing that several Eat (reduced food intake) mutants, especially eat-2, have extended longevity.

Publication: J. Apfeld, C. Kenyon, Cell nonautonomy of C. elegans daf-2 function in the regulation of diapause and life-span. Cell 95, 199-210 (1998).

This paper is the first to apply mosaic analysis to the specification of longevity in C. elegans (some work had been done in Drosophila almost 20 years earlier). Genetic mosaic analysis is a method by which researchers can generate an organism that is made up of two or more distinct genotypes that have been mixed together so that any cell can be of any genotype. The precise cells that control the phenotype of interest can be identified using this method. The paper showed convincingly that dauer formation is controlled by the nervous system in a cell-autonomous manner. It also demonstrated that daf-2 functions in the neuroectoderm, and possibly in other tissues, to regulate life-span. The experiments revealed that daf-2 activity is required in the AB cell lineage to influence life-span. This lineage produces the nervous system, some epidermis, and part of the pharynx. The pharynx can be ruled out as the source of daf-2 function, because the life-span regulatory activity was not localized to the cells that produce the pharynx (ABa). However, alternate interpretations of the longevity data indicating cell non-autonomy cannot be ruled out from the data presented.

Publication: Shin Murakami, Thomas E. Johnson, Life extension and stress resistance in Caenorhabditis elegans modulated by the tkr-1 gene. Curr. Biol. 8, 1091-1094 (1998) [see erratum in Curr. Biol. 9, R791 (1999)].

At this point in history, C. elegans was the only metazoan in which mutations that extend longevity had been identified directly. All of these mutations were either null mutations or gave rise to proteins with reduced function. This paper reported the discovery of a new class of genes that lead to extended longevity when overexpressed. The first gene in this class was designated tyrosine kinase receptor-1 (tkr-1 ) because it bore homology to tyrosine kinase receptors identified in many species. In subsequent papers, tkr-1 is referred to as old-1, for overexpression longevity determinant. Overexpression of old-1 also renders worms resistant to various types of stress (such as heat and ultraviolet irradiation), strengthening the relation between increased stress resistance and life-span extension.

Publication: Y. Honda, S. Honda, The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans. FASEB J. 13, 1385-1393 (1999).

Previously, Larson (1993) and Vanfleteren (1993) showed that the longevity-extending daf-2 mutation also confers resistance to oxidative stress. Here, the authors showed that this so-called "Oxr" phenotype is enhanced by the clk-1 mutation. These findings led the authors to test whether the daf-2 pathway also regulates the expression of genes encoding antioxidant defense enzymes. Indeed, they found that the concentration of mRNA from the sod-3 gene, which encodes Mn-SOD, was much higher in daf-2 mutants than in wild-type worms. In addition, the increase in sod-3 gene expression was regulated by the insulin-like signaling pathway. clk-1 mutant worms did not display the Oxr phenotype; nor did they exhibit an increase in the expression of sod-3. However, worms carrying both the daf-2 and clk-1 mutations had higher amounts of sod-3 expression than did daf-2 mutant worms. These findings suggest (i) that the daf-2 pathway controls longevity, at least in part, by regulating the MnSOD-associated antioxidant defense system, and (ii) that clk-1 is involved indirectly in the daf-2 longevity program.

2000 Back to Top

Publication: D. W. Walker, G. McColl, N. L. Jenkins, J. Harris, G. J. Lithgow, Evolution of life-span in C. elegans. Nature 405, 296-297 (2000).

This is the first paper to address the question of the evolutionary fitness of a gerontogene mutant. A large body of literature in evolutionary genetics "proves" that aging and senescence are not adaptive and are thus emergent properties of the evolutionary process, which cannot eliminate defective mutations that are only expressed after reproduction is complete [for more information on evolutionary biology and aging, see M. R. Rose, Evolutionary Biology of Aging (Oxford University Press, Oxford, 1991)]. Previous studies had failed to find a growth or reproductive disadvantage in age-1 mutants under normal laboratory conditions of abundant food. This paper showed that in an environment that fluctuates between abundant food and starvation (that is, a wild-type environment), a wild-type strain of C. elegans is more fit than animals carrying the age-1 reference allele (hx546). The authors developed a competition methodology in which the mutant and the wild type were grown together on the same plate.

Publication: C. A. Wolkow, K. D. Kimura, M. S. Lee, G. Ruvkun, Regulation of C. elegans life-span by insulin-like signaling in the nervous system. Science 290, 147-150 (2000).

In this report, the authors demonstrated that neuronal expression is necessary and sufficient for dauer formation and longevity extension. The authors used various promoters to drive expression of a wild-type version of daf-2 in various tissues and showed that expression in neuronal cells completely rescued the dauer and longevity phenotypes of daf-2 mutants. The key concept in this paper is that aging and dauer formation are both regulated by the nervous system (see also Apfeld and Kenyon, 1998, above).

Publication: Simon Melov, Joanne Ravenscroft, Sarwatt Malik, Matt S. Gill, David W. Walker, Peter E. Clayton, Douglas C. Wallace, Bernard Malfroy, Susan R. Doctrow, Gordon J. Lithgow, Extension of life-span with superoxide dismutase/catalase mimetics. Science 289, 1567-1569 (2000).

These authors tested the theory that reactive oxygen species cause aging (see "The Two Faces of Oxygen"). To this end, they treated worms with a small-molecule, synthetic antioxidant drug (an SOD/catalase mimetic) and found that life-span was extended dramatically in worms that had consumed the drug. This is the first demonstration of dramatic life-span extension by pharmaceutical intervention.

2001 Back to Top

Publication 1: Heidi A. Tissenbaum, Leonard Guarente, Increased dosage of a sir-2 gene extends life-span in Caenorhabditis elegans. Nature 410, 227-330 (2001).

Publication 2: M. Tatar, A. Kopelman, D. Epstein, M.-P. Tu, C.-M. Yin, R. S. Garofalo, A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 292, 107-110 (2001).

Publication 3: David J. Clancy, David Gems, Lawrence G. Harshman, Sean Oldham, Hugo Stocker, Ernst Hafen, Sally J. Leevers, Linda Partridge, Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein.

These three papers showed that the functions of life-extension genes are conserved across species. The Guarente lab has shown that over-expression of the Saccharomyces cerevisiae gene SIR2 in yeast enhances longevity. Sir2p encodes an nicotinamide adenine dinucleotide-dependent histone deacetylase and is required for life-span extension by caloric restriction in yeast. In the paper above, Tissenbaum and Guarente demonstrated that overexpression of sir-2.1, the worm homolog of yeast SIR2, extends longevity in nematodes. Importantly, they also showed that the extended longevity phenotype from overexpression of sir-2.1 can be suppressed by mutations in daf-16.

The other two papers cited above (publications 2 and 3) showed that the role of insulin/IGF signaling in modulating the life-span of an animal is evolutionarily conserved. The fruit fly Drosophila melanogaster has an insulin-like receptor encoded by the InR gene. InR shares sequence similarity with nematode daf-2, as well as with mammalian genes that encode insulin and IGF-1 receptors. In Tatar et al. above, the authors demonstrated that overexpression of InR yields dwarf flies that display enhanced longevity when compared with wild-type flies. In the related paper above, Clancy et al. describe experiments in flies with chico , a Drosophila gene that encodes an InR substrate. They found that mutations in the chico gene yield flies with an extended life-span.

Publication: S. B. Pierce, M. Costa, R. Wisotzkey, S. Devadhar, S. A. Homburger, A. R. Buchman, K. C. Ferguson, J. Heller, D. M. Platt, A. A. Pasquinelli, L. X. Liu, S. K. Doberstein, G. Ruvkun, Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes Dev. 15, 672-686 (2001).

Pierce et al. create an interspecies model in which human insulin is tested in C. elegans. Overexpression led to results that contradict expectations based on current models of the DAF-2 pathway, but can be interpreted to mean either an evolutionary shift in signal transduction or aberrant interactions in such cross-species in vivo assays.

Publication 1: C. B�nard, B. McCright, Y. Zhang, S. Felkai, B. Lakowski, S. Hekimi, The C. elegans maternal-effect gene clk-2 is essential for embryonic development, encodes a protein homologous to yeast Tel2p and affects telomere length. Development 128, 4045-4055 (2001).]

Publication 2: S. Ahmed, A. Alpi, M. O. Hengartner, A. Gartner, C. elegans RAD-5/CLK-2 defines a new DNA damage checkpoint protein. Curr. Biol. 11, 1934-1944 (2001).

Publication 3: C. A. Lim, I. S. Mian, A. F. Dernburg, J. Campisi, C. elegans clk-2, a gene that limits life-span, encodes a telomere length regulator similar to yeast telomere binding protein Tel2p. Curr Biol. 11, 1706-1710 (2001).

These three papers appeared simultaneously and showed two things. First, they revealed that the previously characterized clk-2 and rad-5 mutations appear in the same gene. clk-2 mutant worms develop slowly as embryos and have a 25% extension of life-span. rad-5 was known to confer heightened sensitivity to DNA-damaging treatments. These results constitute the first indication that clk-2 has a role in monitoring DNA damage. Second, the clk-2/rad-5 gene was shown to encode a protein that is homologous to the yeast protein Tel2p, which participates in the determination of telomere length. For a more detailed discussion of these surprising results, see "How a clk Ticks."

2002 Back to Top

Publication: P. L. Larsen, C. F. Clarke, Extension of life-span in Caenorhabditis elegans by a diet lacking coenzyme Q. Science 295, 120-123 (2002).

A series of studies from this lab has shown that coenzyme Q deficiency probably underlies the slow growth and Age phenotypes of clk-1 mutants. A diet deficient in coenzyme Q creates a Clk phenocopy. It seems that C. elegans is tuned to increase longevity under conditions that probably would kill most mammals. For more information on this interesting finding, see "Eschew Beaucoup Q" .

Publication: Nuno Arantes-Oliveira, Javier Apfeld, Andrew Dillin, Cynthia Kenyon, Regulation of life-span by germ-line stem cells in Caenorhabditis elegans. Science 295, 502-505 (2002).

This study extends several earlier findings from the Kenyon lab showing that enhanced longevity is under both negative and positive control by the germline and somatic tissues of the gonad, respectively. For a more extensive treatment of these findings, see the Tatar Perspective.

Future History Back to Top

2020: The dramatic discoveries on the genetics of longevity made in the 1980s and 90s, first in the nematode and then in yeast and Drosophila, have led to the identification of many mammalian homologs that appear to act in similar manners. Pharmaceutical companies were very reluctant to invest heavily in the area, given the lack of biomarkers for aging and the expected lack of ability to establish a proprietary position. Nevertheless, Elixir and then several other startups successfully developed drugs that target these genes as well as the extensive set of additional regulatory proteins not found in nematodes. These therapeutics are termed "elegogs," for the organism in which antiaging mutants were first found in the 1980s. Clinical trials conducted with these drugs have led the Food and Drug Administration (FDA) to approve several of them for the treatment of various short-term illnesses. However, much off-label use of these drugs is already apparent, particularly among athletes, dancers, actors, and politicians.

2050: The initial phase of illegitimate drug use to curb aging gave way in the 2020s and 2030s to drugs that specifically targeted genes involved in basic mechansims of aging. These drugs have been shown to be efficacious in mammals and to slow one or more aspects of the aging process. As a result of the malicious side effects of the first aging-related drugs, which were approved for use in the 20s (see 2020 Future History), the FDA has vigorously enforced prohibitions against off-label use of subsequent elegogs. Interestingly enough, the early attempts to corner the market by the elegog companies backfired on them when a massive class action suit filed by the majority of the world's 7 billion citizens resulted in a landmark legal finding that established the elegogs as a "World Drug," and resulted in such reduced manufacturing costs that the drugs are now routinely added to most water supplies throughout the world.

12002: For a taste of the future, I refer you to a single provocative Web site. Many more Web sites could be included, but this site references a book, Daughter of Elysium, in which original work on C. elegans is immortalized in various ways as being the first ever to show that aging could be combated, leading ultimately to "immortality."

For other opinions with respect to "future history," see and

For those who wish to add their opinions on future history (or anything else in this review), click on the Comment on Article link, found at the bottom of this review or in the tool bar on the left side of the page.

August 28, 2002 Citation: T. E. Johnson, Subfield History: Caenorhabditis elegans as a System for Analysis of the Genetics of Aging Science's SAGE KE (28 August 2002),;2002/34/re4

JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16.
S. W. Oh, A. Mukhopadhyay, N. Svrzikapa, F. Jiang, R. J. Davis, and H. A. Tissenbaum (2005)
PNAS 102, 4494-4499
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