Kenyon CJ et al. (2000) West Coast Worm Meeting "Expression and regulation of daf-16::gfp constructs"

Kenyon CJ, Lin K-C

[West Coast Worm Meeting, 2000]

Wild-type C.elegans ages rapidly, undergoing development, senescence, and death in less than 3 weeks. In contrast, mutants with reduced activity of daf-2, a homolog of the insulin and insulin-like growth factor (IGF-1) receptors (Kimura et al., 1997), age more slowly and live more than twice as long (Kenyon et al., 1993). Wild-type DAF-2 activity also promotes growth to adulthood and prevents dauer formation, since severe loss of daf-2 function causes the animals to become dauers in the presence of food. Both the lifespan extension and dauer-constitutive phenotypes caused by daf-2 mutations are dependent on the activity of daf-16, which encodes an HNF-3/forkhead family member (Ogg et al., 1997; Lin et al., 1997). We have created GFP-tagged daf-16 constructs and used them to study the expression and regulation of daf-16. We will report our progress on this study.

MJ Munoz et al. (1999) International C. elegans Meeting "Isolating genes asociated with longevity"

DL Riddle, MJ Munoz

[International C. elegans Meeting, 1999]

Dauer larvae can live for as long as six months, whereas the life span of animals that develop continuosly to the adult is only about two weeks. Temperature-sensitive daf-2 mutants enter the dauer stage constitutively at 25degC, and when grown at permissive temperature their adult life span is doubled (Kenyon et al. 1993). Mutations in daf-16 , encoding a forkhead transcription factor (Ogg et al. 1997, Lin et al. 1997), suppress the increased daf-2 longevity. Thus, expression of some of the genes that are targets of DAF-16 activation may be responsible for increased longevity. We are using the differential display PCR method (Liang and Pardee 1997) to look for differences in gene expression between a daf-2 mutant (long life span) and a daf-2; daf-16 double mutant (wild-type life span). We have identified two differentially expressed genes that have a higher level of expression in the daf-2 mutants. The sequence of these clones predicted two different proteins with unknown function. We have also looked for differences in gene expression between a daf-2; daf-12 double mutant (four times wild-type life span) and a daf-2; daf-12; daf-16 triple mutant (wild-type life span) (Larsen et al. 1995). In this case we have found eleven differentially expressed genes. Seven of them are expressed at higher levels in the triple mutant. We have sequenced two of these and both encode proteins homologous to lectins. The other four genes are expressed at higher levels in the daf-2; daf-12 double mutant. We have cloned and sequenced one, which encodes a protein with unknown function. We are cloning and sequencing the remaining genes. We plan to integrate this information with that obtained from other methods (SAGE analysis, subtraction libraries and microarrays) to select candidates for genes that enhance longevity. We then plan test the effect of their (1) overexpression and (2) loss of function on life span. Kenyon et al. (1993) Nature 366:461-464 Larsen P. et al. (1995) Genetics 139:1567-1583 Liang P. and Pardee, A. B. (1997) Science 257:967-971 Lin K et al. (1997) Science 278:1319-1322 Ogg et al. (1997) Nature 389:994-999

Munoz MJ et al. (1998) Midwest Worm Meeting "ISOLATING GENES INVOLVED IN C.ELEGANS LIFE SPAN."

Munoz MJ, Riddle DL

[Midwest Worm Meeting, 1998]

Dauer larvae can live for as long as six months, whereas the maximum life span of animals that develop continuously to the adult is only about three weeks. Temperature-sensitive daf-2 mutants enter the dauer stage at 25!C even in conditions that are good for growth and reproduction. When the mutants develop at 15!C they do not become dauer larvae, but their adult life span is doubled (Kenyon et al. Nature 366: 461- 464). This effect has been interpreted as indicating that even at the permissive temperature, these nematodes have activated part of a dauer "efficient life maintenance" program. Mutations in daf-16, encoding a fork head transcription factor (Ogg et al. Nature 389: 994-999, Lin et al. Science 278: 1319-1322), suppress the increase in adult life span of the daf-2 mutant. Thus, expression of a particular set of genes (targets of DAF-16 activation) may be responsible for increased longevity. We are using the differential display PCR method to look for differences in gene expression between daf-2 mutants (long life span) and the daf-2;daf-16 double mutant (wild-type life span). Differentially expressed genes will be cloned and tested for the effect of their expression on life span.

Gupta, Pratyush et al. (2013) International Worm Meeting "MRG-1 and RFP-1 regulate proliferation in the germline."

Jasper, Katie, Gupta, Pratyush, Hansen, David, Leahul, Lindsay

[International Worm Meeting, 2013]

We previously demonstrated that the proteasome inhibits the proliferative fate in the C. elegans germ line. Reduced proteasomal function enhances over-proliferation in a sensitized genetic background (Macdonald, Knox et al. 2008). Presumably, the proteasome is involved in degrading proteins that either promote the proliferative fate, or inhibit meiotic entry. We refer to these as proliferation promoting proteins (PPPs). To identify these PPPs, we first sought to identify the substrate recognition subunits (SRS) of E3 ubiquitin ligases that may target the PPPs for degradation by the proteasome. We screened 826 SRSs by RNAi in four sensitized genetic backgrounds and found five that enhance over-proliferation. One of these, RFP-1, specifically enhances glp-1(ar202gf) to form a germline tumor, albeit incomplete. Large-scale 2-hybrid screens identified potential binding partners for RFP-1 (Crowe and Candido 2004; Zhong and Sternberg 2006), which could be PPPs that are targeted for proteasomal degradation by RFP-1. One of these, mrg-1, partially suppresses the over-proliferation phenotype of glp-1(ar202gf); rfp-1(ok572), suggesting that it may function as a PPP. We found that MRG-1 levels do increase in an rfp-1 mutant, as well as when proteasomal function is decreased through genetic mutation or chemical inhibition. Therefore, MRG-1 protein levels are likely regulated through proteasomal-mediated degradation. We are currently determining how mrg-1 may participate in controlling the proliferative fate in the germline.

K Lin et al. (1999) International C. elegans Meeting "isolation OF ADDITIONAL DAF-2 SUPPRESSORS"

C Kenyon, K Lin

[International C. elegans Meeting, 1999]

The wild-type C.elegans ages rapidly, undergoing development, senescence, and death in less then 3 weeks. In contrast, mutants with reduced activity of daf-2 , a homolog of the insulin and insulin-like growth factor (IGF-1) receptors (Kimura et al., 1997), age more slowly and live more than twice as long (Kenyon et al., 1993). Wild-type DAF-2 activity also promotes growth to adulthood and prevents dauer formation, since severe loss of daf-2 function causes the animals to become dauers in the presence of food. Both the lifespan extension and dauer-constitutive phenotypes caused by daf-2 mutations are dependent on the activity of daf-16 , which encodes an HNF-3/forkhead family member (Ogg et al., 1997; Lin et al., 1997). Previous screens for suppressors of the dauer-constitutive phenotype of daf-2 in our lab have resulted in isolation of just additional alleles of daf-16 (Lin et al., 1997). In order to overcome this problem and isolate additional daf-2 suppressors, we created daf-2(e1370) animals with extra copies of the wild-type daf-16 gene, by injecting a rescuing genomic daf-16 construct and integrating the arrays into the genome. The integrated array fully rescued dauer formation in the daf-16(null);daf-2(e1370) background. We screened a total of 100,000 genomes for suppressors of the dauer constitutive phenotype of daf-2(e1370) , and we're now screening for suppressors of the lifespan extension phenotype. We will report our progress in characterizing the suppressors we isolated.

H Hsin et al. (1999) International C. elegans Meeting "Signals from the Reproductive System that Regulate the Lifespan of C. elegans"

CJ Kenyon, H Hsin

[International C. elegans Meeting, 1999]

Understanding how the aging process can be regulated is a fascinating and fundamental problem in biology. We have found that signals from the reproductive system influence the lifespan of the nematode C. elegans . If the cells that give rise to the germline are killed with a laser microbeam, the lifespan of the animal is extended (Hsin and Kenyon, Nature , in press). Our findings suggest that germline signals act by modulating the activity of an insulin/IGF-1-like pathway known to regulate the aging of this organism. Mutants with reduced activity of the insulin/IGF-1 receptor homolog DAF-2 have been shown to live twice as long as normal (1,2,3), and their longevity requires the activity of DAF-16, a member of the forkhead/winged-helix family of transcriptional regulators (1,2,4,5). We find that in order for germline ablation to extend lifespan, DAF-16 is required, as well as a putative nuclear hormone receptor, DAF-12 (6,7). In addition, our findings suggest that signals from the somatic gonad have an opposite effect on lifespan, and that this effect appears to require DAF-2 activity. Together our findings imply that the C. elegans insulin/IGF-1 system integrates multiple signals to define the rate of aging of the animal. This study demonstrates an inherent relationship between the reproductive state of this animal and its lifespan, and may have implications for the co-evolution of reproductive capability and longevity. 1. C. Kenyon, et al., Nature 366 , 461-4 (1993). 2. P. Larsen, P. Albert, D. Riddle, Genetics 139 , 1567-83 (1995). 3. K. Kimura, H. Tissenbaum, Y. Liu, G. Ruvkun, Science 277 , 942-6 (1997). 4. K. Lin, J. Dorman, A. Rodan, C. Kenyon, Science 278 , 1319-22 (1997). 5. S. Ogg, et al., Nature 389 , 994-9 (1997). 6. W-H. Yeh. Ph.D. Thesis, University of Missouri, Columbia (1991). 7. D. Riddle, P. Albert, in C. elegans II D. Riddle, T. Blumenthal, B. Meyer, J. Priess, Eds. (Cold Spring Harbor Laboratory Press, 1997) pp. 739-68.

R Lee et al. (1999) International C. elegans Meeting "Extension of C. elegans lifespan by a mutation in daf-21/hsp9"

R Lee, G Ruvkun

[International C. elegans Meeting, 1999]

Longevity often associates with resistance to environmental stress. In C. elegans , long-lived (Age) mutants such as age-1 and daf-2 are more resistant to oxidative, UV-radiation, or thermal stresses. To get at the proximal causes for this longevity-associated stress resistance, we are focusing on the worm stress-response gene hsp90 because it is a molecular chaperone; in contrast to many other 'house-keeping' genes, its mRNA level is dramatically increased in the dauer compared to other developmental stages (Dalley & Golomb 1992, Dev Biol 151: 80). The Thomas lab found that daf-21(p673) has a missense mutation in the worm hsp90 gene (Malone and Thomas, WBG14.4: 52). daf-21(p673) has a temperature-sensitive dauer constitutive (Daf-c) phenotype. Based on this phenotype, genetic analysis showed that p673 is a recessive, possibly neomorphic mutation that encourages dauer formation by affecting chemosensory neuron function (Malone et al, IWM93: 297). We found that daf-21(p673) mutant lived about 40-50% longer (mean and maximal adult lifespan) than wild-type animals when shifted from the permissive (15deg) to the restrictive (25deg) temperature at the L4 or young adult stages. Whereas either daf-16(mgDf47) or daf-12(m20) efficiently suppressed the Daf-c phenotype (Thomas et al 1993, Genetics 134: 1105), only daf-16 suppressed Age, suggesting that daf-21 may affect dauer formation and lifespan independantly. daf-16 is a member of the Forkhead class of transcription factors; and it mediates all of the effects caused by daf-2 insulin-like signaling, including lifespan (Ogg et al 1997, Nature 389: 994; Lin et al 1997, Science 278: 1319). One possible explanation for the observation that a daf-16 null mutation is epistatic to daf-21 is that daf-21 expression is positively regulated by daf-16 . We are testing this hypothesis. We are also asking if Age mutants in general have an elevated hsp90 expression or function. We thank Jim Thomas for providing daf-21 mutant strains.

SB Pierce et al. (1999) International C. elegans Meeting "ins-14, one of many insulin-related genes in C.elegans, can regulate dauer formation"

L Liu, R Wisotsky, SB Pierce, G Ruvkun

[International C. elegans Meeting, 1999]

The activity of daf-2 , which encodes a homolog of insulin and insulin-like growth factor I (IGF-I) receptors (1), is required for non-dauer development and normal adult life span, and is likely to be regulated by one or more insulin-like ligands. By searching the C. elegans genome database, we identified 30 ins genes predicted to encode insulin-like peptides, that could be grouped into several subfamilies on the basis of structural similarities. One gene, ins-14 on F13B12, contains likely C-peptide cleavage sites typical of mammalian insulins. To investigate whether this family of insulin-related genes could play a role in dauer formation, several members were overexpressed by injecting worms with the appropriate PCR-amplified genomic regions. We hypothesized that overexpression of putative DAF-2 ligands might suppress the Daf-c phenotype of weak daf-2 mutants or mutants in the parallel daf-7 TGF-ß pathway, which synergizes with the daf-2 insulin-like signaling pathway (2). None of the tested genes suppressed daf-2(e1365) , a non-null allele, although these transgenic worms may not have produced enough ligand to overcome the impaired DAF-2 receptor. However, the ins-14 , but not the M04D8.2 or T10D8.a&b, transgene maternally suppressed the Daf-c phenotype of daf-7(e1372) . Similar results were seen with an ins-14 transgene in which the coding region was replaced with the cDNA for human insulin. An ins-14::GFP transgene is expressed in many neurons, intestine, and vulval muscles. Finally, we isolated a deletion mutant, ins-14(nr2091) , in which the entire coding region is removed. ins-14(nr2091) had no dauer phenotype as a single mutant, but enhanced the Daf-c phenotype of daf-7 in the ins-14(nr2091);daf-7(e1372) double mutant. The suppression of the daf-7(e1372) Daf-c phenotype by ins-14 overexpression and its enhancement by the ins-14(nr2091) mutation is consistent with ins-14 acting as a DAF-2 agonist. Other members of the ins gene family may act either redundantly as agonists or possibly as antagonists of DAF-2. (1) Kimura et al, Science 1997;277:942 (2) Ogg et al, Nature 1997;389:994

John J Thaden et al. (1999) International C. elegans Meeting "Effects on nitrogen and energy metabolism of a defective DAF-2 insulin receptor-like protein"

Daniel Owen, Heather S Reis, Henry J Mroczkowski, Kewen Jauss, Robert J Shmookler Reis, John J Thaden

[International C. elegans Meeting, 1999]

The insulin/IGF-1 receptor homolog DAF-21 must function in order for C. elegans to develop, reproduce, and age normally 2 (unless the forkhead transcription factor DAF-16 also is defective 3 ). A proline-to-serine mutation e1370ts in the DAF-2 kinase domain1 causes constitutive dauer formation in homozygotes grown at 25 C, and ~100% life extension in adults upshifted at L4 4 . Unlike dauers, daf-2 adults feed and reproduce, but like dauers, they do accumulate excess fat, suggesting that daf-2 regulates metabolism in adults 1 . Might the DAF longevity phenotype be due to a metabolic equivalent of dietary restriction? That daf-2 double mutants with pharyngeal pumping defects have life spans further extended 5 implies otherwise. Here, we report that CB1370 daf-2(e1370) incorporated as much tritium as N2 and JT7098 daf-2;daf-16(m26) when fed for 1h on OP50 previously grown with 3H-amino acids, whereas JT5488 daf-2;daf-12(m20) incorporated less. Also, life spans of both CB1370 daf-2 and JT7098 daf-2;daf-16 were extended 30% by dietary restriction. These results support the conclusion 5 that DAF-2 longevity is not due to a genetically-induced caloric restriction. In spite of similar feeding, excretion of ammonia nitrogen was 61-67% less for four daf-2 strains, compared to a panel of other strains. In progress are measurements of excreted amino acids, apparently the other major nitrogen end product for nematodes. Reduced ammonia excretion indicates that nitrogen metabolism is also under daf-2 regulation. It may be that proteins and purines, like fats, are stored in large excess by dauer larvae and daf-2 adults; however, the latter was not evident in measures of nematode total protein. Supported by NIH grant AG-09413. 1. Kimura et al. (1997) Science 277, 942-8. 2. Gems et al. (1998) Genetics 150, 129-55. 3. Ogg et al. (1997) Nature 389, 994-9; and Lin et al. (1997) Science 278, 1319-22. 4. Kenyon et al. (1993) Nature 366, 461-4; and Larsen et al. (1995) Genetics 139, 1567-83. 5. Lakowski & Hekimi (1998) Proc Nat Acad Sci USA 95, 13091-6.

Garth McGrath et al. (2003) International Worm Meeting "daf-18/PTEN Functions in Part of the DNA Damage Response"

Mike Costa, Damian Curtis, Garth McGrath

[International Worm Meeting, 2003]

The phosphoinositide phosphatase PTEN is a tumor suppressor gene that is commonly mutated in a variety of human cancer types, and loss of PTEN function is associated with increased cell survival and proliferation. We have isolated new mutants for the C. elegans PTEN ortholog daf-18 and characterized their phenotype with respect to processes related to tumor biology. In a forward mutagenesis screen designed to identify daf-18 loss-of-function alleles, we screened 10,000 haploid genomes for mutants that maternally suppress the daf-2 dauer-constitutive mutant phenotype, a characteristic previously reported for daf-18 mutants (Gil et al, 1999). Two mutants were isolated from this screen and both had a moderately penetrant ruptured vulva phenotype, another phenotype associated with daf-18 mutations (Ogg and Ruvkun, 1998). By sequencing, we identified a daf-18 nonsense mutation in one of the mutants and a daf-18 missense mutation in the other mutant, and find that these mutations co-segregate with both the maternal daf-2 suppression and ruptured vulva phenotypes. The role of daf-18 in dauer formation has been well characterized, and we wanted to determine if daf-18 mutants have additional phenotypes that might be related to the tumor formation resulting from PTEN loss in humans. We approached this by examining the daf-18 mutants for defects in the DNA damage response, including DNA damage-induced germline apoptosis, mitotic cell cycle arrest, and reduced fecundity. We found that daf-18 mutants appear to have normal apoptotic and mitotic cell cycle arrest responses in the germline, but appear hypersensitive for the reduced fecundity in response to irradiation at the L1 stage. The hypersensitivity to radiation-induced fecundity defects seem to result from both decreased germline proliferation and reduced progeny viability. A deletion mutant for the DNA damage checkpoint gene cep-1/p53 is only slightly hypersensitive for radiation-induced fecundity defects, and it synergistically enhances daf-18 mutants to a level of hypersensitivity similar to that of a rad-5 checkpoint mutant. One possibility is that this phenotype may arise from a defect in DNA repair, and that the DNA damage sensors (including RAD-5) signal in parallel to both DAF-18 and CEP-1 which, in turn, activate partially overlapping sets of DNA repair genes.