Apfeld J et al. (1996) West Coast Worm Meeting "A GENETIC SCREEN FOR LONG-LIVED WORMS."

Hsin H, Dorman JB, Kenyon CJ, Apfeld J, Albinder B

[West Coast Worm Meeting, 1996]

We are interested in finding genes that control lifespan in C. elegans. We are currently doing a clonal screen for mutations that lead to an increase in lifespan. We mutagenize a temperature-sensitive sterile strain, clone out F2 progeny, allow these worms to self fertilize, clone some of their progeny into a new plate at the restrictive temperature, and screen these for increased lifespan. So far we have screened 360 F2 clones and are in the process of retesting 3 of them. Javier Apfeld is also studying the gene daf-2. The e1370 allele of this gene has multiple phenotypes: at 25°C daf-2(e1370) worms develop into dauer larvae (a developmentally arrested, stress resistant, long lived alternative larval form); at 20°C daf-2(e1370) worms do not form dauer larvae, but live 2.5 times longer than wild-type (N2) worms. Javier wants to know where daf-2 functions and how it acts, and is currently doing mosaic analysis to address these questions.

Apfeld J et al. (1997) International C. elegans Meeting "daf-2 Controls Dauer Formation Non-Autonomously"

Apfeld J, Kenyon CJ

[International C. elegans Meeting, 1997]

Mutations in daf-2 lead to a doubling of both mean and maximum lifespan. This gene also represses the developmental decision to form a dauer larva. Is daf-2 required within individual tissues to promote their normal development? Or does daf-2 function in some cells to control the development of other cells? Also, does daf-2 function in the same set of cells to regulate both dauer formation and lifespan? To answer these questions we are analyzing daf-2 genetic mosaics. We have found two classes of mosaics. Mosaic animals that lack daf-2 activity in the AB descendants (i.e., AB mosaics) form full dauers. In addition, some ABa mosaics also form full dauers; in these worms, daf-2(+) cells, such as the hypodermal seam cells V3 and V5, have a dauer phenotype. On the other hand, P1 and MS mosaics develop into normal looking fertile adults. In these worms daf-2(-) cells have a non-dauer phenotype; for example, the daf-2(-) sex myoblasts generate functional egg-laying muscles. Our results suggest that daf-2 is necessary and sufficient within the AB descendants to prevent dauer formation. Additionally, we find that the dauer phenotype of some cells, such as the hypodermal seam cells and sex myoblasts, is controlled by the daf-2 genotype of other cells. We are continuing the mosaic analysis in order to investigate where daf-2 function is required to regulate lifespan.

Apfeld J et al. (1998) West Coast Worm Meeting "CELL NON-AUTONOMY OF daf-2 FUNCTION IN THE REGULATION OF LIFESPAN AND DAUER FORMATION"

Kenyon CJ, Apfeld J

[West Coast Worm Meeting, 1998]

Mutations in daf-2, a homologue of the insulin and insulin-like growth factor-I (IGF-I) receptors, can lead to a doubling of both mean and maximum lifespan. This gene also regulates dauer diapause. Is daf-2 activity required within each individual cell to promote its growth into adulthood and to regulate its lifespan? Or does this gene function within signaling cells that, in turn, control the fates of individual cells in the animal? We used genetic mosaic analysis of the daf-2 gene to distinguish between these possibilities. We found that daf-2 acts cell non-autonomously to regulate lifespan extension, entry and exit from dauer diapause. Furthermore, the pattern of genetic mosaics that we observed indicates that the site where daf-2 activity is required to control these processes is composed of cells with partially redundant activities that arise from multiple branches of the C. elegans cell lineage. We propose that daf-2 activity controls the activity of endocrine signals that act on individual tissues to regulate adult lifespan and dauer formation.

J Apfeld et al. (1999) International C. elegans Meeting "Multiple Layers of Cell Non-autonomy in the Control of C. elegans Lifespan"

C Kenyon, J Apfeld

[International C. elegans Meeting, 1999]

The insulin/IGF-receptor homologue daf-2 regulates aging in C. elegans . Decreasing daf-2 activity causes fertile adults to remain active much longer than normal and to live more than twice as long. A more severe decrease in daf-2 function causes young larvae to enter a state of diapause rather than progressing to adulthood. The lifespan extension and dauer formation phenotypes observed in daf-2 mutants are dependent on the activity of daf-16 , a transcription factor of the HNF-3/winged-helix family. We have asked which cells require daf-2 gene activity in order for the animal to develop to adulthood and to age normally. We found that daf-2 functions cell non-autonomously in both processes. Our findings imply that the lifespan of C. elegans is determined by a signaling cascade in which daf-2 acts in multiple cell lineages to regulate the production or activity of a secondary signal (or signals), which, in turn, controls the growth and longevity of individual tissues in the animal. C. elegans obtains information about its external environment through sensory cilia. We have found that animals with defective sensory cilia live longer than animals with intact sensory cilia. This lifespan extension is dependent on the activity of daf-16 , and mutations that disrupt cilium structure do not further increase the lifespan of daf-2 mutants. The long-lived cilium structure mutants feed normally, suggesting that sensory perception itself, rather than an associated effect on food intake, influences lifespan. The lifespan of C. elegans is also under the control of signals from the germline; laser ablation of the germline precursors leads to a lifespan extension that is dependent on the activity of daf-16 (1). We have found that a number of mutations that interfere in different ways with the development of the germline also extend lifespan. Together, our results indicate that the C. elegans insulin/IGF-1 like signaling pathway may integrate two different types of information in setting the lifespan of the animal: internal signals that reflect the state of the germline, and external signals that may convey information about the animal's surrounding environment. (1) Hsin and Kenyon, Nature, in press.

Wolfgang Maier et al. (2008) European Worm Meeting "A putative neuropeptide receptor that senses food quality to inhibit longevity"

Martin Regenass, Bakhtiyor Adilov, Wolfgang Maier, Joy Alcedo

[European Worm Meeting, 2008]

The lifespan of C. elegans is influenced by its sensory system [1, 2], but. the mechanisms that underlie this sensory influence remain largely unknown.. The genome of the worm encodes a large number of predicted neuropeptide. receptors, which may modulate the information processed by the sensory. system and thus affect lifespan. Consistent with this hypothesis, we have. found that nmur-1, a gene encoding a receptor with homology to mammalian. neuromedin U receptors (NMURs), inhibits longevity. An nmur-1::GFP. reporter construct is expressed in neurons, including sensory neurons, and. the somatic gonad.. nmur-1 appears to act with the worm''s insulin/IGF-1 receptor, daf-2, to. inhibit longevity, but functions independent of the FOXO transcription. factor daf-16, which acts downstream of daf-2. In addition, unlike. mutations in many longevity-inhibiting genes, including daf-2, the deletion. of nmur-1 causes only subtle phenotypes in processes that require energy. expenditure. This suggests that nmur-1 plays a minor role in regulating. the overall metabolism of the worm. Interestingly, the lifespan phenotype. of nmur-1 is dependent on its bacterial food source. Thus, nmur-1 appears. to be involved in sensing food quality to influence lifespan.. References: [1] Apfeld, J., and C. Kenyon. 1999. Nature 402: 804 - 809.. [2] Alcedo, J., and C. Kenyon. 2004. Neuron 41: 45 - 55.

Schiffer, Jodie et al. (2017) International Worm Meeting "How do worms determine their readiness to cope with environmental stress?"

Stumbur, Stephanie, Heath, William, Tam, Hannah, McGowan, Natalie, Vogelaar, Abigail, Schiffer, Jodie, Apfeld, Javier, Stanley, Julian

[International Worm Meeting, 2017]

At any time in their life, worms may encounter harmful environmental conditions such as high concentrations of oxidants or high temperature. We are interested in understanding how worms prepare for these potentially lethal events. We want to know: what establishes how prepared they are? To determine the mechanisms that control survival under harmful conditions, we are examining the effects of strong loss-of-function and null mutants in a collection of intercellular signaling receptors and transcription factors regulated by these receptors. We measure survival under oxidative stress and at high temperature using a "Lifespan Machine" cluster of flat-bed scanners [1]. This automated technology presents substantial advancements in throughput and sensitivity compared to manual methods. Using this approach, we have identified several signaling receptors and transcription factors that regulate survival under oxidative conditions. Interestingly, we identified both receptors and transcription factors that function to either confer or limit oxidative-stress resistance. This indicates that the combined level of activity of these genes sets the worm's readiness to cope with oxidative stress. We are currently investigating whether these genes act together or independently to determine the worm's normal level of resistance to various stressors, and the mechanisms that establish the normal activity levels of each of these genetic determinants of stress resistance. Reference: 1. Stroustrup N, Ulmschneider BE, Nash ZM, Lopez-Moyado IF, Apfeld J, Fontana W. The Caenorhabditis elegans Lifespan Machine. Nature Methods. 2013;10(7):665-70.

Michelle Keaney et al. (2001) International C. elegans Meeting "Optimising dietary restriction-mediated lifespan extension"

Michelle Keaney, David Gems

[International C. elegans Meeting, 2001]

A number of reports have shown that dietary restriction (DR) results in increased lifespan in C. elegans, with the magnitude of increase depending upon the method used (1-4). How DR extends lifespan is unknown, but one possibility is that it down-regulates the insulin/IGF (IIS) signalling pathway which regulates ageing. Work to date is both consistent with (5) and contradictory to (4) this hypothesis. We are testing four methods used previously to exert DR: diluted monoxenic liquid culture (1), reduced nutrient NGM (2), axenic culture (3), and the use of eat mutations (4). We are determining which method can be optimised to (a) give the greatest magnitude of lifespan extension; (b) can be made quantitative (so the degree of DR imposed can be varied); and (c) is easy to perform. We are also seeking to maximise DR-mediated lifespan extension such that further reduction of dietary intake would lead to malnutrition and premature death. Our aim is to use an optimised DR protocol to understand the mode of action of DR, and its relationship to IIS, reproduction and oxidative damage. (1) Klass, M.R. Mech. Ageing Dev. 6 : 413 (1977). (2) Hosono, R. et al. Exp. Geront. 24 : 251 (1989). (3) Vanfleteren, J. R. and De Vreese, A. FASEB J. 9 : 1355 (1995). (4) Lakowski, B. and Hekimi, S. PNAS USA 95 : 13091 (1998). (5) Apfeld, J. and Kenyon, C. Nature 402 : 804 (1999).

Wolfgang Maier et al. (2006) European Worm Meeting "Identification of Genes Involved in the Neural Regulation of Lifespan"

Martin Regenass, Joy Alcedo, Wolfgang Maier, Monique Thomas

[European Worm Meeting, 2006]

Wolfgang Maier, Martin Regenass, Monique Thomas and Joy Alcedo. Aging is influenced by several factors, which include the environment of the animal. For example, mutations that impair sensory function extend the lifespan of C. elegans, which suggests that lifespan, of at least this animal, is regulated by the perception of environmental cues (1). Specific sensory neurons appear to regulate lifespan, at least in part, through the insulin/IGF-1 pathway (1, 2). Since several putative insulin-like ligands are also expressed in these neurons (3), it is possible that an environmental signal(s) sensed by these neurons regulates the production of these ligands. To elucidate further the molecular mechanisms of the sensory influence on lifespan, we are carrying out an RNA-mediated interference screen for enhancers and suppressors of the lifespan phenotype of sensory mutant animals. As we identify genes involved in the sensory influence on lifespan, we will study their expression patterns using GFP-fusion reporter constructs to determine in which cells they function. We also plan to measure the lifespans of strains in which the particular gene was knocked out or overexpressed. Furthermore, we will conduct pertinent studies on how the gene products function, such as testing if the functions of these genes require each other or involve the daf-2 insulin/IGF-1 pathway or other signaling pathways in the worm known to influence its lifespan. References: (1) Apfeld, J., and C. Kenyon. 1999. Nature (Lond.). 402: 804 809; (2) Alcedo, J., and C. Kenyon. 2004. Neuron 41: 45 55; (3) Pierce, S. B., et al. 2001. Genes Dev. 15: 672 - 686.

Alcedo J et al. (2000) West Coast Worm Meeting "Temporal and spatial requirement of sensory cilia in the regulation of worm lifespan"

Apfeld J, Kenyon CJ, Hsin H, Dorman JB, Tsung BT, Albinder B, Alcedo J

[West Coast Worm Meeting, 2000]

C. elegans is an excellent organism in which to study the regulation of lifespan. Genetic analyses have already shown that an insulin/IGF-like hormonal control system, the DAF-2 pathway, regulates lifespan in the worm. However, many components of the pathway, whose existence has been inferred from previous studies (Apfeld and Kenyon, (1999). Nature 402, 804-809; and Hsin and Kenyon, (1999) Nature 399, 362-366) have not been identified. To search for more genes that would help to elucidate mechanisms that control lifespan in the worm, our group has done an EMS mutagenesis screen for long-lived worms and found 29 independent long-lived mutants. At least one of the mutants, mu377(IV), maps to and fails to complement the gene daf-10, which is required for the normal development of the worms sensory cilia. Apfeld and Kenyon (1999) have recently shown that worms defective in sensory cilia, including daf-10 mutants, live longer than normal worms, which suggests that the lifespan of C. elegans might be regulated by the perception of a signal(s) from the environment. mu377(IV) has a temperature-sensitive defect in its sensory cilia, which is linked to its temperature-sensitive lifespan phenotype. At the restrictive temperature, its lifespan is longer than wild-type and it exhibits a defect in its sensory cilia; whereas at the permissive temperature, its lifespan is the same as wild-type and it exhibits normal sensory cilia. Since the defect in the sensory cilia of mu377(IV) is reversible, we determined the temporal requirement for the gene product of mu377(IV) in regulating the lifespan of the worm. To gain insight into the spatial requirement for sensory perception in controlling the worms lifespan, we are also in the process of determining which sensory neurons are necessary to regulate its lifespan by expressing wild-type sensory genes in subsets of mutant sensory neurons.

Stanley, J et al. (2019) International Worm Meeting "What is your genetically-encoded biosensor good for?"

Stanley, J, Apfeld, J

[International Worm Meeting, 2019]

Genetically-encoded biosensors have revolutionized our ability to measure a wide variety of cellular properties in live animals. As experimentalists, any time a new sensor is developed, we would like to know: what is that sensor good for? That is, what range of values of the cellular property of interest is that sensor well-suited to measure accurately? Here, we present a theoretical framework to determine the suitability of biosensors with two states. Two-state biosensors are simple sensors that change conformation, and spectral properties, in response to a specific input. Existing two-state biosensors respond to a wide variety of important cellular properties, including pH, ATP, and glutathione redox potential. In our previous work with the roGFP1_R12 sensor, we deployed a mathematical framework that enabled us to calculate glutathione redox potential from fluorescence ratio measurements given knowledge of the spectral and biochemical properties of the sensor, and the properties of our microscope. We extended this framework to analyze how the precision of microscopy measurements limits the accuracy of calculated glutathione redox potentials. This enabled us to predict the range of redox potentials that roGFP1_R12 is well-suited to measure given a theoretical measurement error and, importantly, with our empirically-determined measurement error. This analysis demonstrated that our sensor is well-suited to measure cytosolic glutathione redox potential in the feeding muscles of live C. elegans over a wide range of environmental and genetic conditions. Next, we applied our theoretical framework to 10 glutathione redox potential sensors with known spectral and biochemical properties. This enabled us to define the range of potentials for which each sensor is well-suited and choose optimal sensors for different applications. Finally, we generalized our framework to all ratiometric two-state biosensors and applied this analysis to families of pH and nucleotide-specific biosensors. We are currently building web-based, interactive tools and documentation to help the community find biosensors that are well-suited for their experiments.