Positive selection of Caenorhabditis elegans mutants with increased stress resistance and longevity

We developed selective conditions for long-lived mutants of the nematode Caenorhabditis elegans by subjecting the first larval stage (L1) to thermal stress at 30 degrees for 7 days. The surviving larvae developed to fertile adults after the temperature was shifted to 15 degrees. A total of one million F(2) progeny and a half million F(3) progeny of ethyl-methanesulfonate-mutagenized animals were treated in three separate experiments. Among the 81 putative mutants that recovered and matured to the reproductive adult, 63 retested as thermotolerant and 49 (80%) exhibited a >15% increase in mean life span. All the known classes of dauer formation (Daf) mutant that affect longevity were found, including six new alleles of daf-2, and a unique temperature-sensitive, dauer-constitutive allele of age-1. Alleles of dyf-2 and unc-13 were isolated, and mutants of unc-18, a gene that interacts with unc-13, were also found to be long lived. Thirteen additional mutations define at least four new genes.     

pkc-1 regulates daf-2 insulin/IGF signalling-dependent control of dauer formation in Caenorhabditis elegans

In Caenorhabditis elegans, the insulin/IGF pathway participates in the decision to initiate dauer development. Dauer is a diapause stage that is triggered by environmental stresses, such as a lack of nutrients. Insulin/IGF receptor mutants arrest constitutively in dauer, an effect that can be suppressed by mutations in other elements of the insulin/IGF pathway or by a reduction in the activity of the nuclear hormone receptor daf‐12. We have isolated a pkc‐1 mutant that acts as a novel suppressor of the dauer phenotypes caused by insulin/IGF receptor mutations. Interactions between insulin/IGF mutants and the pkc‐1 suppressor mutant are similar to those described for daf‐12 or the DAF‐12 coregulator din‐1. Moreover, we show that the expression of the DAF‐12 target daf‐9, which is normally elevated upon a reduction in insulin/IGF receptor activity, is suppressed in a pkc‐1 mutant background, suggesting that pkc‐1 could link the daf‐12 and insulin/IGF pathways. pkc‐1 has been implicated in the regulation of peptide neurosecretion in C. elegans. Although we demonstrate that pkc‐1 expression in the nervous system regulates dauer formation, our results suggest that the requirement for pkc‐1 in neurosecretion is independent of its role in modulating insulin/IGF signalling. pkc‐1 belongs to the novel protein kinase C (nPKC) family, members of which have been implicated in insulin resistance and diabetes in mammals, suggesting a conserved role for pkc‐1 in the regulation of the insulin/IGF pathway.     

The longevity of Caenorhabditis elegans in soil

Relatively simple model organisms such as yeast, fruit-flies and the nematode, Caenorhabditis elegans, have proven to be invaluable resources in biological studies. An example is the widespread use of C. elegans to investigate the complex process of ageing. An important issue when interpreting results from these studies is the similarity of the observed C. elegans mortality pattern in the laboratory to that expected in its natural environment. We found that the longevity of C. elegans under more natural conditions is reduced up to 10-fold compared with standard laboratory culture conditions. Additionally, C. elegans mutants that live twice as long as wild-type worms in laboratory conditions typically die sooner than wild-type worms in a natural soil. These results indicate that conclusions regarding extended longevity drawn from standard laboratory assays may not extend to animals in their native environment.     

The L-isoaspartyl-O-methyltransferase in Caenorhabditis elegans larval longevity and autophagy

The protein L-isoaspartyl-O-methyltransferase, coded by the pcm-1 gene in Caenorhabditis elegans, participates in the repair of age-damaged proteins. We tested the ability of pcm-1-deficient nematodes to survive starvation stress as developmentally-arrested L1 larvae. We found that pcm-1 mutant L1 larvae do not survive as well as wild-type L1 larvae when incubated in M9 medium without nutrients. We then tested whether the starved L1 larvae could continue development when allowed access to food in a recovery assay. A loss of recovery ability with age was observed for all larvae, with little or no difference between the pcm-1 mutant and wild-type N2 larvae. Interestingly, when L1 larvae were starved in cholesterol-containing S medium or M9 medium supplemented with cholesterol, the survival rates of both mutant and wild-type animals nearly doubles, with pcm-1 larvae again faring more poorly than N2 larvae. Furthermore, L1 larvae cultured in these cholesterol-containing media show an increase in Sudan Black staining over animals cultured in M9 medium. The longevity defects of pcm-1 mutants previously seen in dauer larvae and here in L1 larvae suggest a defect in the ability of pcm-1 mutants to recycle and reuse old cellular components in pathways such as autophagy. Using an autophagosomal marker, we found evidence suggesting that the pcm-1 mutation may inhibit autophagy during dauer formation, suggesting that the absence of protein repair may also interfere with protein degradation pathways.     

Uncoupling of longevity and paraquat resistance in mutants of the nematode Caenorhabditis elegans

To analyze the relationship between resistance to oxidative stress and longevity, we isolated three novel paraquat-resistant mutants, mev-5, mev-6, and mev-7, from the nematode Caenorhabditis elegans. They all showed the Dyf (defective in dye filling) phenotype, but not always resistance to heat or UV. Life-span extension was observed only in the mev-5 mutant at 26 degrees C. These results indicate that longevity is uncoupled with the phenotype of paraquat resistance.     

A network of stimulatory and inhibitory Galpha-subunits regulates olfaction in Caenorhabditis elegans

The two pairs of sensory neurons of C. elegans, AWA and AWC, that mediate odorant attraction, express six Galpha-subunits, suggesting that olfaction is regulated by a complex signaling network. Here, we describe the cellular localization and functions of the six olfactory Galpha-subunits: GPA-2, GPA-3, GPA-5, GPA-6, GPA-13, and ODR-3. All except GPA-6 localize to sensory cilia, suggesting a direct role in sensory transduction. GPA-2, GPA-3, GPA-5, and GPA-6 are also present in cell bodies and axons and GPA-5 specifically localizes to synaptic sites. Analysis of animals with single- to sixfold loss-of-function mutations shows that olfaction involves a balance between multiple stimulatory and inhibitory signals. ODR-3 constitutes the main stimulatory signal and is sufficient for the detection of odorants. GPA-3 forms a second stimulatory signal in the AWA and AWC neurons, also sufficient for odorant detection. In AWA, signaling is suppressed by GPA-5. In AWC, GPA-2 and GPA-13 negatively and positively regulate signaling, respectively. Finally, we show that only ODR-3 plays a role in cilia morphogenesis. Defects in this process are, however, independent of olfactory behavior. Our findings reveal the existence of a complex signaling network that controls odorant detection by C. elegans.     

The Caenorhabditis elegans ect-2 RhoGEF gene regulates cytokinesis and migration of epidermal P cells

A reduction-of-function mutation in ect-2 was isolated as a suppressor of the Multivulva phenotype of a lin-31 mutation. Analysis using markers indicates that this mutation causes defects in both the cytokinesis and migration of epidermal P cells, phenotypes similar to those caused by expressing a rho-1 dominant-negative construct. ect-2 encodes the Caenorhabditis elegans orthologue of the mouse Ect2 and Drosophila Pebble that function as guanine nucleotide exchange factors (GEFs) for Rho GTPases. The ect-2Colon, two colonsGFP reporter is expressed in embryonic cells and P cells. RNA interference of ect-2 causes sterility and embryonic lethality, in addition to the P-cell defects. We have determined the lesions of two ect-2 alleles, and described their differences in phenotypes in specific tissues. We propose a model in which ECT-2GEF not only activates RHO-1 for P-cell cytokinesis, but also collaborates with UNC-73GEF and at least two Rac proteins to regulate P-cell migration.     

X-linked gene expression and sex determination in Caenorhabditis elegans

The signal for sex determination in the nematode Caenorhabditis elegans is the ratio between the number of X chromosomes and the number of sets of autosomes (the X/A ratio). Animals with an X/A ratio of 0.67 (a triploid with two X chromosomes) or less are males. Animals with an X/A ratio of 0.75 or more are hermaphrodites. Thus, diploid males have one X chromosome and diploid hermaphrodites have two X chromosomes. However, the difference in X-chromosome number between the sexes is not reflected in general levels of X-linked gene expression because of the phenomenon of dosage compensation. In dosage compensation, X-linked gene expression appears to be 'turned down' in 2X animals to the 1X level of expression. An intriguing and unexplained finding is that mutations and X-chromosome duplications that elevate X-linked gene expression also feminize triploid males. One way that this relationship between sex determination and X-linked gene expression may be operating is discussed.     

The Caenorhabditis elegans PcG-like gene sop-2 regulates the temporal and sexual specificities of cell fates

How spatial, temporal, and sexual specific cues are integrated to specify distinct cell fates during multicellular organism development is largely unknown. Here we demonstrate that the Caenorhabditis elegans PcG-like gene sop-2 determines the temporal and sexual specificities of a row of hypodermal seam cells, in addition to specifying their positional identities. Loss-of-function of sop-2 causes premature expression of adult fates at larval stages. sop-2 acts upstream of lin-29 in the heterochronic pathway and genetically interacts with other heterochronic genes in specifying the temporal fates of seam cells at different larval stages. We show that the number of ALG-1-containing P bodies is increased in seam cells in sop-2 mutants. Furthermore, the microRNA-mediated repression of a heterochronic gene reporter is enhanced in sop-2 mutants. Mutations in sop-2 also cause partial hermaphrodite-to-male sexual transformations. The homeotic transformations, heterochronic defects, and sexual transformations can occur concomitantly in sop-2 mutants. In summary, our studies reveal that sop-2 integrates spatial, temporal, and sexual cues during C. elegans development.     

daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans.

Evolutionary models of aging propose that a trade-off exists between the resources an organism devotes to reproduction and growth and those devoted to cellular maintenance and repair, such that an optimal life history always entails an imperfect ability to resist stress. Yet, since environmental stressors, such as caloric restriction or exposure to mild stress, can increase stress resistance and life span, it is possible that a common genetic mechanism could regulate the allocation of resources in response to a changing environment (for overview, see ). Consistent with predictions of evolutionary trade-off models, we show that nematodes carrying an integrated DAF-16::GFP transgene grow and reproduce more slowly yet are more stress resistant and longer lived than controls carrying the integration marker alone. We also show that the nuclear localization of the DAF-16::GFP fusion protein responds to environmental inputs as well as genetic. Environmental stresses, such as starvation, heat, and oxidative stress, cause rapid nuclear localization of DAF-16. In conditions rich in food, we find that DAF-16::GFP is inhibited from entry into the nucleus by daf-2 and akt-1/akt-2, both components of insulin-like signaling in nematodes. We suggest that changes in the subcellular localization of DAF-16 by environmental cues allows for rapid reallocation of resources in response to a changing environment at all stages of life.     

The divergent orphan nuclear receptor ODR-7 regulates olfactory neuron gene expression via multiple mechanisms in Caenorhabditis elegans

Nuclear receptors regulate numerous critical biological processes. The C. elegans genome is predicted to encode approximately 270 nuclear receptors of which >250 are unique to nematodes. ODR-7 is the only member of this large divergent family whose functions have been defined genetically. ODR-7 is expressed in the AWA olfactory neurons and specifies AWA sensory identity by promoting the expression of AWA-specific signaling genes and repressing the expression of an AWC-specific olfactory receptor gene. To elucidate the molecular mechanisms of action of a divergent nuclear receptor, we have identified residues and domains required for different aspects of ODR-7 function in vivo. ODR-7 utilizes an unexpected diversity of mechanisms to regulate the expression of different sets of target genes. Moreover, these mechanisms are distinct in normal and heterologous cellular contexts. The odr-7 ortholog in the closely related nematode C. briggsae can fully substitute for all ODR-7-mediated functions, indicating conservation of function across 25-120 million years of divergence.