Daf-2, Daf-16 and Daf-23: Genetically Interacting Genes Controlling Dauer Formation in Caenorhabditis Elegans

Under conditions of high population density and low food, Caenorhabditis elegans forms an alternative third larval stage, called the dauer stage, which is resistant to desiccation and harsh environments. Genetic analysis of some dauer constitutive (Daf-c) and dauer defective (Daf-d) mutants has revealed a complex pathway that is likely to function in particular neurons and/or responding tissues. Here we analyze the genetic interactions between three genes which comprise a branch of the dauer formation pathway that acts in parallel to or downstream of the other branches of the pathway, the Daf-c genes daf-2 and daf-23 and the Daf-d gene daf-16. Unlike mutations in other Daf-c genes, mutations in both daf-2 and daf-23 cause non-conditional arrest at the dauer stage. Our epistasis analysis suggests that daf-2 and daf-23 are functioning at a similar point in the dauer pathway. First, mutations in daf-2 and daf-23 are epistatic to mutations in the same set of Daf-d genes. Second, daf-2 and daf-23 mutants are suppressed by mutations in daf-16. Mutations in daf-16 do not suppress any of the other Daf-c mutants as efficiently as they suppress daf-2 and daf-23 mutants. Third, double mutants between either daf-2 or daf-23 and several other daf-d mutants exhibit an unusual interaction. Based on these results, we present a model for the function of daf-2, daf-23 and daf-16 in dauer formation.     

Genetic Analysis of the Roles of Daf-28 and Age-1 in Regulating Caenorhabditis Elegans Dauer Formation

Based on environmental cues, the nervous system of Caenorhabditis elegans regulates formation of the dauer larva, an alternative larval form specialized for long-term survival under harsh conditions. Mutations that cause constitutive or defective dauer formation (Daf-c or Daf-d) have been identified and the genes ordered in a branched pathway. Most Daf-c mutations also affect recovery from the dauer stage. The semi-dominant mutation daf-28(sa191) is Daf-c but has no apparent effect on dauer recovery. We use this unique aspect of daf-28(sa191) to characterize the effects of several Daf-d and synthetic Daf-c mutations on dauer recovery. We present double mutant analysis that indicates that daf-28(sa191) acts at a novel point downstream in the genetic pathway for dauer formation. We also show that daf-28(sa191) causes a modest increase (12-13%) in life span. The phenotypes and genetic interactions of daf-28(sa191) are most similar to those of daf-2 and daf-23 mutations, which also cause a dramatic increase in life span. We present mapping and complementation data that suggest that daf-23 is the same gene as age-1, identified previously by mutations that extend life span. We find that age-1 alleles are also Daf-c at 27°.     

An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans.

Mutations in daf-2 and age-1 cause a dramatic increase in longevity as well as developmental arrest at the dauer diapause stage in Caenorhabditis elegans. daf-2 and age-1 encode components of an insulin-like signaling pathway. Both daf-2 and age-1 act at a similar point in the genetic epistasis pathway for dauer arrest and longevity and regulate the activity of the daf-16 gene. Mutations in daf-16 cause a dauer-defective phenotype and are epistatic to the diapause arrest and life span extension phenotypes of daf-2 and age-1 mutants. Here we show that mutations in this pathway also affect fertility and embryonic development. Weak daf-2 alleles, and maternally rescued age-1 alleles that cause life span extension but do not arrest at the dauer stage, also reduce fertility and viability. We find that age-1(hx546) has reduced both maternal and zygotic age-1 activity. daf-16 mutations suppress all of the daf-2 and age-1 phenotypes, including dauer arrest, life span extension, reduced fertility, and viability defects. These data show that insulin signaling, mediated by DAF-2 through the AGE-1 phosphatidylinositol-3-OH kinase, regulates reproduction and embryonic development, as well as dauer diapause and life span, and that DAF-16 transduces these signals. The regulation of fertility, life span, and metabolism by an insulin-like signaling pathway is similar to the endocrine regulation of metabolism and fertility by mammalian insulin signaling.     

The Age-1 and Daf-2 Genes Function in a Common Pathway to Control the Lifespan of Caenorhabditis Elegans

Recessive mutations in two genes, daf-2 and age-1, extend the lifespan of Caenorhabditis elegans significantly. The daf-2 gene also regulates formation of an alternative developmental state called the dauer. Here we asked whether these two genes function in the same or different lifespan pathways. We found that the longevity of both age-1 and daf-2 mutants requires the activities of the same two genes, daf-16 and daf-18. In addition, the daf-2(e1370); age-1(hx546) double mutant did not live significantly longer than the daf-2 single mutant. We also found that, like daf-2 mutations, the age-1(hx546) mutation affects certain aspects of dauer formation. These findings suggest that age-1 and daf-2 mutations do act in the same lifespan pathway and extend lifespan by triggering similar if not identical processes.     

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.     

LET-60 RAS modulates effects of insulin/IGF-1 signaling on development and aging in Caenorhabditis elegans

The DAF-2 insulin/insulin-like growth factor 1 (IGF-1) receptor signals via a phosphatidylinositol 3-kinase (PI3K) pathway to control dauer larva formation and adult longevity in Caenorhabditis elegans. Yet epistasis analysis suggests signal bifurcation downstream of DAF-2. We have used epistasis analysis to test whether the Ras pathway (which plays a role in signaling from mammalian insulin receptors) acts downstream of DAF-2. We find that an activated Ras mutation, let-60(n1046gf), weakly suppresses constitutive dauer diapause in daf-2 and age-1 (PI3K) mutants. Moreover, increased Ras pathway signaling partially suppresses the daf-2 mutant feeding defect, while reduced Ras pathway signaling enhances it. By contrast, activated Ras extends the longevity induced by mutation of daf-2, while reduced Ras pathway signaling partially suppresses it. Thus, Ras pathway signaling appears to act with insulin/IGF-1 signaling during larval development, but against it during aging.     

NCR-1 and NCR-2, the C. elegans homologs of the human Niemann-Pick type C1 disease protein, function upstream of DAF-9 in the dauer formation pathways

Mutations in the human NPC1 gene cause most cases of Niemann-Pick type C (NP-C) disease, a fatal autosomal recessive neurodegenerative disorder. NPC1 is implicated in intracellular trafficking of cholesterol and glycolipids, but its exact function remains unclear. The C. elegans genome contains two homologs of NPC1, ncr-1 and ncr-2, and an ncr-2; ncr-1 double deletion mutant forms dauer larvae constitutively (Daf-c). We have analyzed the phenotypes of ncr single and double mutants in detail, and determined the ncr gene expression patterns. We find that the ncr genes function in a hormonal branch of the dauer formation pathway upstream of daf-9 and daf-12, which encode a cytochrome P450 enzyme and a nuclear hormone receptor, respectively. ncr-1 is expressed broadly in tissues with high levels of cholesterol, whereas expression of ncr-2 is restricted to a few cells. Both Ncr genes are expressed in the XXX cells, which are implicated in regulating dauer formation via the daf-9 pathway. Only the ncr-1 mutant is hypersensitive to cholesterol deprivation and to progesterone, an inhibitor of intracellular cholesterol trafficking. Our results support the hypothesis that ncr-1 and ncr-2 are involved in intracellular cholesterol processing in C. elegans, and that a sterol-signaling defect is responsible for the Daf-c phenotype of the ncr-2; ncr-1 mutant.     

A Genetic Pathway Conferring Life Extension and Resistance to Uv Stress in Caenorhabditis Elegans

A variety of mechanisms have been proposed to explain the extension of adult life span (Age) seen in several mutants in Caenorhabditis elegans (age-1: an altered aging rate; daf-2 and daf-23: activation of a dauer-specific longevity program; spe-26: reduced fertility; clk-1: an altered biological clock). Using an assay for ultraviolet (UV) resistance in young adult hermaphrodites (survival after UV irradiation), we observed that all these Age mutants show increased resistance to UV. Moreover, mutations in daf-16 suppressed the UV resistance as well as the increased longevity of all the Age mutants. In contrast to the multiple mechanisms initially proposed, these results suggest that a single, daf-16-dependent pathway, specifies both extended life span and increased UV resistance. The mutations in daf-16 did not alter the reduced fertility of spe-26 and interestingly a daf-16 mutant is more fertile than wild type. We propose that life span and some aspects of stress resistance are jointly negatively regulated by a set of gerontogenes (genes whose alteration causes life extension) in C. elegans.     

Intercellular signaling of reproductive development by the C. elegans DAF-9 cytochrome P450

Parallel pathways control C. elegans reproductive development in response to environmental cues. Attenuation of daf-2 insulin-like or daf-7 TGFbeta-like signaling pathways cause developmental arrest at the stress resistant and long-lived dauer stage. Loss-of-function mutations in the cytochrome P450 gene daf-9 also cause dauer arrest and defects in cell migration. A rescuing daf-9::GFP fusion gene driven by the daf-9 promoter is expressed in two head cells at all stages, in the hypodermis from mid-second larval stage (L2) to the fourth larval stage (L4), and in the spermatheca of the adult hermaphrodite. Although the level of daf-9::GFP expression in the head cells and spermatheca is constant, hypodermal daf-9::GFP expression is modulated by multiple inputs. In particular, daf-9::GFP expression in the hypodermis is absolutely dependent on daf-12, the nuclear receptor that is negatively regulated by daf-9 gene activity, suggesting feedback control between daf-9 and daf-12 in this tissue. daf-9 expression exclusively in the hypodermis is sufficient to restore reproductive development in daf-9 mutant animals, suggesting that daf-9 functions in a cell nonautonomous manner. Furthermore, constitutive expression of daf-9 in the hypodermis suppresses dauer arrest of daf-7 mutant animals and inhibits dauer remodelling of some tissues in daf-2 mutant animals. Thus, daf-9 may integrate outputs from daf-2 and daf-7 signaling pathways to relay neuroendocrine signals through synthesis of a lipophilic hormone.     

A Screen for Nonconditional Dauer-Constitutive Mutations in Caenorhabditis Elegans

In Caenorhabditis elegans, formation of the developmentally arrested dauer larva is induced by high levels of a constitutively secreted pheromone. Synergy between two groups of incompletely penetrant dauer-constitutive (Daf-c) mutations has recently led to a proposal that these two groups of genes are partially redundant and function in two parallel pathways that regulate dauer formation. A possible weakness in this reasoning is that the mutations used to identify the synergy were specifically obtained as incompletely penetrant mutations. Here we use screens to identify new Daf-c alleles without any requirement for partial penetrance. Nevertheless, 22 of the 25 new mutations are incompletely penetrant mutations in 6 previously identified genes. Among these are mutations in daf-8 and daf-19, genes for which only one mutation had been previously identified. Also included in this group are three daf-1 alleles that do not exhibit the maternal rescue characteristic of other daf-1 alleles. Two of the 25 new mutations are fully penetrant and are alleles of daf-2, the one gene in which a fully penetrant mutation had been found earlier. Finally, one of the 25 new mutations is semidominant, temperature-sensitive, and identifies a new gene, daf-28. The results demonstrate that an incompletely penetrant Daf-c phenotype is characteristic of mutations in most Daf-c genes other than daf-2. This finding strengthens the hypothesis that a branched genetic pathway controls dauer formation.     

The thioredoxin TRX-1 modulates the function of the insulin-like neuropeptide DAF-28 during dauer formation in Caenorhabditis elegans

Thioredoxins comprise a conserved family of redox regulators involved in many biological processes, including stress resistance and aging. We report that the C. elegans thioredoxin TRX-1 acts in ASJ head sensory neurons as a novel modulator of the insulin-like neuropeptide DAF-28 during dauer formation. We show that increased formation of stress-resistant, long-lived dauer larvae in mutants for the gene encoding the insulin-like neuropeptide DAF-28 requires TRX-1 acting in ASJ neurons, upstream of the insulin-like receptor DAF-2. Genetic rescue experiments demonstrate that redox-independent functions of TRX-1 specifically in ASJ neurons are needed for the dauer formation constitutive (Daf-c) phenotype of daf-28 mutants. GFP reporters of trx-1 and daf-28 show opposing expression patterns in dauers (i.e. trx-1 is up-regulated and daf-28 is down-regulated), an effect that is not observed in growing L2/L3 larvae. In addition, functional TRX-1 is required for the down-regulation of a GFP reporter of daf-28 during dauer formation, a process that is likely subject to DAF-28-mediated feedback regulation. Our findings demonstrate that TRX-1 modulates DAF-28 signaling by contributing to the down-regulation of daf-28 expression during dauer formation. We propose that TRX-1 acts as a fluctuating neuronal signaling modulator within ASJ neurons to monitor the adjustment of neuropeptide expression, including insulin-like proteins, during dauer formation in response to adverse environmental conditions.     

Lipophilic regulator of a developmental switch in Caenorhabditis elegans

Abstract In Caenorhabditis elegans, the decision to develop into a reproductive adult or arrest as a dauer larva is influenced by multiple pathways including insulin-like and transforming growth factor beta (TGFbeta)-like signalling pathways. It has been proposed that lipophilic hormones act downstream of these pathways to regulate dauer formation. One likely target for such a hormone is DAF-12, an orphan nuclear hormone receptor that mediates these developmental decisions and also influences adult lifespan. In order to find lipophilic hormones we have generated lipophilic extracts from mass cultures of C. elegans and shown that they rescue the dauer constitutive phenotype of class 1 daf-2 insulin signalling mutants and the TGFbeta signalling mutant daf-7. These extracts are also able to rescue the lethal dauer phenotype of daf-9 mutants, which lack a P450 steroid hydroxylase thought to be involved in the synthesis of the DAF-12 ligand; extracts, however, have no effect on a DAF-12 ligand binding domain mutant that is predicted to be ligand insensitive. The production of this hormone appears to be DAF-9 dependent as extracts from a daf-9;daf-12 double mutant do not exhibit this activity. Preliminary fractionation of the lipophilic extracts shows that the activity is hydrophobic with some polar properties, consistent with a small lipophilic hormone. We propose that the dauer rescuing activity is a hormone synthesized by DAF-9 that acts through DAF-12.     

Control of DAF-7 TGF-(alpha) expression and neuronal process development by a receptor tyrosine kinase KIN-8 in Caenorhabditis elegans.

KIN-8 in C. elegans is highly homologous to human ROR-1 and 2 receptor tyrosine kinases of unknown functions. These kinases belong to a new subfamily related to the Trk subfamily. A kin-8 promoter::gfp fusion gene was expressed in ASI and many other neurons as well as in pharyngeal and head muscles. A kin-8 deletion mutant was isolated and showed constitutive dauer larva formation (Daf-c) phenotype: about half of the F(1) progeny became dauer larvae when they were cultivated on an old lawn of E. coli as food. Among the cells expressing kin-8::gfp, only ASI sensory neurons are known to express DAF-7 TGF-(beta), a key molecule preventing dauer larva formation. In the kin-8 deletion mutant, expression of daf-7::gfp in ASI was greatly reduced, dye-filling in ASI was specifically lost and ASI sensory processes did not completely extend into the amphid pore. The Daf-c phenotype was suppressed by daf-7 cDNA expression or a daf-3 null mutation. ASI-directed expression of kin-8 cDNA under the daf-7 promoter or expression by a heat shock promoter rescued the dye-filling defect, but not the Daf-c phenotype, of the kin-8 mutant. These results show that the kin-8 mutation causes the Daf-c phenotype through reduction of the daf-7 gene expression and that KIN-8 function is cell-autonomous for the dye-filling in ASI. KIN-8 is required for the process development of ASI, and also involved in promotion of daf-7 expression through a physiological or developmental function.     

Evidence for Parallel Processing of Sensory Information Controlling Dauer Formation in Caenorhabditis Elegans

Dauer formation in Caenorhabditis elegans is induced by chemosensation of high levels of a constitutively secreted pheromone. Seven genes defined by mutations that confer a dauer-formation constitutive phenotype (Daf-c) can be congruently divided into two groups by any of three criteria. Group 1 genes (daf-11 and daf-21) are (1) strongly synergistic with group 2 genes for their Daf-c phenotype, (2) incompletely suppressed by dauer-formation defective (Daf-d) mutations in the genes daf-3 and daf-5 and (3) strongly suppressed by Daf-d mutations in nine genes that affect the structure of chemosensory endings. Group 2 genes (daf-1, daf-4, daf-7, daf-8 and daf-14) are (1) strongly synergistic with group 1 genes for their Daf-c phenotype, (2) fully suppressed by Daf-d mutations in daf-3 and daf-5 and (3) not suppressed by Daf-d mutations in the nine genes that affect chemosensory ending structure. Mutations in each group of genes also cause distinct additional behavioral defects. We propose that these two groups of Daf-c genes act in parallel pathways that process sensory information. The two pathways are partially redundant with each other and normally act in concert to control dauer formation.