Steroids as central regulators of organismal development and lifespan

Larvae of the nematode Caenorhabditis elegans must choose between reproductive development and dauer diapause. This decision is based on sensing of environmental inputs and dauer pheromone, a small molecule signal that serves to monitor population density. These signals are integrated via conserved neuroendocrine pathways that converge on steroidal ligands of the nuclear receptor DAF-12, a homolog of the mammalian vitamin D receptor and liver X receptor. DAF-12 acts as the main switch between gene expression programs that drive either reproductive development or dauer entry. Extensive studies in the past two decades demonstrated that biosynthesis of two bile acid-like DAF-12 ligands, named dafachronic acids (DA), controls developmental fate. In this issue of PLoS Biology, Wollam et al. showed that a conserved steroid-modifying enzyme, DHS-16, introduces a key feature in the structures of the DAF-12 ligands, closing a major gap in the DA biosynthesis pathway. The emerging picture of DA biosynthesis in C. elegans enables us to address a key question in the field: how are complex environmental signals integrated to enforce binary, organism-wide decisions on developmental fate? Schaedel et al. demonstrated that pheromone and DA serve as competing signals, and that a positive feedback loop based on regulation of DA biosynthesis ensures organism-wide commitment to reproductive development. Considering that many components of DA signaling are highly conserved, ongoing studies in C. elegans may reveal new aspects of bile acid function and lifespan regulation in mammals. C. elegans normally goes through a simple life cycle: from egg, through four larval stages, to reproductive adult. However, under adverse environmental conditions, these worms enter an alternate third larval stage termed dauer. Compared to normal third stage larvae, dauer larvae have dramatically different metabolism and physiology, and distinct morphology and behavior [1], which confer greatly increased stress resistance and facilitate dispersal. When environmental conditions improve, C. elegans exit dauer and resume reproductive development. The dauer stage is generally considered as “non-aging,” as dauers can persist for months before recovering to develop into a reproductive adult that lives the normal lifespan of a few weeks. Not surprisingly, recent findings suggest that re-activation of some of the molecular signature of dauer later in life contributes to prolonged longevity in C. elegans [2].     

Neuronal KGB-1 JNK MAPK signaling regulates the dauer developmental decision in response to environmental stress in Caenorhabditis elegans

In response to stressful growth conditions of high population density, food scarcity, and elevated temperature, young larvae of nematode Caenorhabditis elegans can enter a developmentally arrested stage called dauer that is characterized by dramatic anatomic and metabolic remodeling. Genetic analysis of dauer formation of C. elegans has served as an experimental paradigm for the identification and characterization of conserved neuroendocrine signaling pathways. Here, we report the identification and characterization of a conserved c-Jun N-terminal Kinase-like mitogen-activated protein kinase (MAPK) pathway that is required for dauer formation in response to environmental stressors. We observed that loss-of-function mutations in the MLK-1-MEK-1-KGB-1 MAPK pathway suppress dauer entry. A loss-of-function mutation in the VHP-1 MAPK phosphatase, a negative regulator of KGB-1 signaling, results in constitutive dauer formation, which is dependent on the presence of dauer pheromone but independent of diminished food levels or elevated temperatures. Our data suggest that the KGB-1 pathway acts in the sensory neurons, in parallel to established insulin and TGF-β signaling pathways, to transduce the dauer-inducing environmental cues of diminished food levels and elevated temperature.     

Daumone fed late in life improves survival and reduces hepatic inflammation and fibrosis in mice

The liver is one of the most susceptible organs to aging, and hepatic inflammation and fibrosis increase with age. Chronic inflammation has been proposed as the major molecular mechanism underlying aging and age-related diseases, whereas calorie restriction has been shown to be the most effective in extending mammalian lifespan and to have anti-aging effects through its anti-inflammatory action. Thus, it is necessary to develop effective calorie restriction mimetics. Daumone [(2)-(6R)-(3,5-dihydroxy-6-methyltetrahydropyran-2-yloxy)heptanoic acid], a pheromone secreted by Caenorhabditis elegans, forces them to enter the dauer stage when facing inadequate conditions. Because Caenorhabditis elegans live longer during the dauer stage under energy deprivation, it was hypothesized that daumone may improve survival in mammals by mimicking calorie restriction. Daumone (2 mg kg⁻¹ day⁻¹) was administered orally for 5 months to 24-month-old male C57BL/6J mice. Daumone was found to reduce the risk of death by 48% compared with age-matched control mice, and the increased plasma insulin normally presented in old mice was significantly reduced by daumone. The increased hepatic hypertrophy, senescence-associated β-galactosidase activity, insulin resistance, lipid accumulation, inflammation, oxidative stress, and fibrosis in old mice were significantly attenuated by daumone. From a mechanistic view, daumone reduced the phosphorylation of the IκBα and upregulation of Rela and Nfkbia mRNA in the livers of old mice. The anti-inflammatory effect of daumone was confirmed in lipopolysaccharide-induced liver injury model. Oral administration of daumone improves survival in mice and delivers anti-aging effects to the aged liver by modulating chronic inflammation, indicating that daumone could be developed as an anti-aging compound.     

Regulation of Dauer Formation by O-GlcNAcylation in Caenorhabditis elegans

Modification of proteins at serine or threonine residues with N-acetylglucosamine, termed O-GlcNAcylation, plays an important role in most eukaryotic cells. To understand the molecular mechanism by which O-GlcNAcylation regulates the entry of Caenorhabditis elegans into the non-aging dauer state, we performed proteomic studies using two mutant strains: the O-GlcNAc transferase-deficient ogt-1(ok430) strain and the O-GlcNAcase-defective oga-1(ok1207) strain. In the presence of the dauer pheromone daumone, ogt-1 showed suppression of dauer formation, whereas oga-1 exhibited enhancement of dauer formation. Consistent with these findings, treatment of wild-type N2 worms with low concentrations of daumone and the O-GlcNAcase inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) enhanced dauer formation, which was dependent on intact O-GlcNAcylation metabolism. We also found that the treatment of daumone enhanced O-GlcNAcylation in vivo. Seven proteins, identified by coupled two-dimensional electrophoresis/liquid chromatography-mass spectroscopy (LC-MS) analysis, were differentially expressed in oga-1(ok1207) worms compared with wild-type N2 worms. The identities of these proteins suggest that O- GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. Using O-GlcNAc labeling with fluorescent dye combined with two-dimensional electrophoresis/LC-MS analysis, we also identified five proteins that were differentially O-GlcNAcylated during dauer formation. Analysis of these candidate O-GlcNAcylated proteins suggests that O-GlcNAcylation may regulate cytoskeleton modifications and protein turnover during dauer formation.     

Divergent evolution of arrested development in the dauer stage of Caenorhabditis elegans and the infective stage of Heterodera glycines

Background

The soybean cyst nematode Heterodera glycines is the most important parasite in soybean production worldwide. A comprehensive analysis of large-scale gene expression changes throughout the development of plant-parasitic nematodes has been lacking to date.

Results

We report an extensive genomic analysis of H. glycines, beginning with the generation of 20,100 expressed sequence tags (ESTs). In-depth analysis of these ESTs plus approximately 1,900 previously published sequences predicted 6,860 unique H. glycines genes and allowed a classification by function using InterProScan. Expression profiling of all 6,860 genes throughout the H. glycines life cycle was undertaken using the Affymetrix Soybean Genome Array GeneChip. Our data sets and results represent a comprehensive resource for molecular studies of H. glycines. Demonstrating the power of this resource, we were able to address whether arrested development in the Caenorhabditis elegans dauer larva and the H. glycines infective second-stage juvenile (J2) exhibits shared gene expression profiles. We determined that the gene expression profiles associated with the C. elegans dauer pathway are not uniformly conserved in H. glycines and that the expression profiles of genes for metabolic enzymes of C. elegans dauer larvae and H. glycines infective J2 are dissimilar.

Conclusion

Our results indicate that hallmark gene expression patterns and metabolism features are not shared in the developmentally arrested life stages of C. elegans and H. glycines, suggesting that developmental arrest in these two nematode species has undergone more divergent evolution than previously thought and pointing to the need for detailed genomic analyses of individual parasite species.     

To Grow or Not to Grow: Nutritional Control of Development During Caenorhabditis elegans L1 Arrest

It is widely appreciated that larvae of the nematode Caenorhabditis elegans arrest development by forming dauer larvae in response to multiple unfavorable environmental conditions. C. elegans larvae can also reversibly arrest development earlier, during the first larval stage (L1), in response to starvation. “L1 arrest” (also known as “L1 diapause”) occurs without morphological modification but is accompanied by increased stress resistance. Caloric restriction and periodic fasting can extend adult lifespan, and developmental models are critical to understanding how the animal is buffered from fluctuations in nutrient availability, impacting lifespan. L1 arrest provides an opportunity to study nutritional control of development. Given its relevance to aging, diabetes, obesity and cancer, interest in L1 arrest is increasing, and signaling pathways and gene regulatory mechanisms controlling arrest and recovery have been characterized. Insulin-like signaling is a critical regulator, and it is modified by and acts through microRNAs. DAF-18/PTEN, AMP-activated kinase and fatty acid biosynthesis are also involved. The nervous system, epidermis, and intestine contribute systemically to regulation of arrest, but cell-autonomous signaling likely contributes to regulation in the germline. A relatively small number of genes affecting starvation survival during L1 arrest are known, and many of them also affect adult lifespan, reflecting a common genetic basis ripe for exploration. mRNA expression is well characterized during arrest, recovery, and normal L1 development, providing a metazoan model for nutritional control of gene expression. In particular, post-recruitment regulation of RNA polymerase II is under nutritional control, potentially contributing to a rapid and coordinated response to feeding. The phenomenology of L1 arrest will be reviewed, as well as regulation of developmental arrest and starvation survival by various signaling pathways and gene regulatory mechanisms.     

Contribution of the Peroxisomal acox Gene to the Dynamic Balance of Daumone Production in Caenorhabditis elegans

Dauer pheromones or daumones, which are signaling molecules that interrupt development and reproduction (dauer larvae) during unfavorable growth conditions, are essential for cellular homeostasis in Caenorhabditis elegans. According to earlier studies, dauer larva formation in strain N2 is enhanced by a temperature increase, suggesting the involvement of a temperature-dependent component in dauer pheromone biosynthesis or sensing. Several naturally occurring daumone analogs (e.g. daumones 1–3) have been identified, and these molecules are predicted to be synthesized in different physiological settings in this nematode. To elucidate the molecular regulatory system that may influence the dynamic balance of specific daumone production in response to sudden temperature changes, we characterized the peroxisomal acox gene encoding acyl-CoA oxidase, which is predicted to catalyze the first reaction during biosynthesis of the fatty acid component of daumones. Using acox-1(ok2257) mutants and a new, robust analytical method, we quantified the three most abundant daumones in worm bodies and showed that acox likely contributes to the dynamic production of various quantities of three different daumones in response to temperature increase, changes that are critical in C. elegans for coping with the natural environmental changes it faces.     

Alteration in cellular acetylcholine influences dauer formation in Caenorhabditis elegans

Altered acetylcholine (Ach) homeostasis is associated with loss of viability in flies, developmental defects in mice, and cognitive deficits in human. Here, we assessed the importance of Ach in Caenorhabditis elegans development, focusing on the role of Ach during dauer formation. We found that dauer formation was disturbed in choline acetyltransferase (cha-1) and acetylcholinesterase (ace) mutants defective in Ach biosynthesis and degradation, respectively. When examined the potential role of G-proteins in dauer formation, goa-1 and egl-30 mutant worms, expressing mutated versions of mammalian G_o and G_q homolog, respectively, showed some abnormalities in dauer formation. Using quantitative mass spectrometry, we also found that dauer larvae had lower Ach content than did reproductively grown larvae. In addition, a proteomic analysis of acetylcholinesterase mutant worms, which have excessive levels of Ach, showed differential expression of metabolic genes. Collectively, these results indicate that alterations in Ach release may influence dauer formation in C. elegans. [BMB Reports 2014; 47(2): 80-85]     

New Tools for Investigating the Comparative Biology of Caenorhabditis briggsae and C. elegans

Comparative studies of Caenorhabditis briggsae and C. elegans have provided insights into gene function and developmental control in both organisms. C. elegans is a well developed model organism with a variety of molecular and genetic tools to study gene functions. In contrast, there are only very limited tools available for its closest relative, C. briggsae. To take advantage of the full potential of this comparative approach, we have developed several genetic and molecular tools to facilitate functional analysis in C. briggsae. First, we designed and implemented an SNP-based oligonucleotide microarray for rapid mapping of genetic mutants in C. briggsae. Second, we generated a mutagenized frozen library to permit the isolation of targeted deletions and used the library to recover a deletion mutant of cbr-unc-119 for use as a transgenic marker. Third, we used the cbr-unc-119 mutant in ballistic transformation and generated fluorescently labeled strains that allow automated lineaging and cellular resolution expression analysis. Finally, we demonstrated the potential of automated lineaging by profiling expression of egl-5, hlh-1, and pha-4 at cellular resolution and by detailed phenotyping of the perturbations on the Wnt signaling pathway. These additions to the experimental toolkit for C. briggsae should greatly increase its utility in comparative studies with C. elegans. With the emerging sequence of nematode species more closely related to C. briggsae, these tools may open novel avenues of experimentation in C. briggsae itself.     

The dauer hypothesis and the evolution of parasitism: 20 years on and still going strong

How any complex trait has evolved is a fascinating question, yet the evolution of parasitism among the nematodes is arguably one of the most arresting. How did free-living nematodes cross that seemingly insurmountable evolutionary chasm between soil dwelling and survival inside another organism? Which of the many finely honed responses to the varied and harsh environments of free-living nematodes provided the material upon which natural selection could act? Although several complementary theories explain this phenomenon, I will focus on the dauer hypothesis. The dauer hypothesis posits that the arrested third-stage dauer larvae of free-living nematodes such as Caenorhabditis elegans are, due to their many physiological similarities with infective third-stage larvae of parasitic nematodes, a pre-adaptation to parasitism. If so, then a logical extension of this hypothesis is that the molecular pathways which control entry into and recovery from dauer formation by free-living nematodes in response to environmental cues have been co-opted to control the processes of infective larval arrest and activation in parasitic nematodes. The molecular machinery that controls dauer entry and exit is present in a wide range of parasitic nematodes. However, the developmental outputs of the different pathways are both conserved and divergent, not only between populations of C. elegans or between C. elegans and parasitic nematodes but also between different species of parasitic nematodes. Thus the picture that emerges is more nuanced than originally predicted and may provide insights into the evolution of such an interesting and complex trait.     

Genetic mapping of variation in dauer larvae development in growing populations of Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the appropriate induction of dauer larvae development within growing populations is likely to be a primary determinant of genotypic fitness. The underlying genetic architecture of natural genetic variation in dauer formation has, however, not been thoroughly investigated. Here, we report extensive natural genetic variation in dauer larvae development within growing populations across multiple wild isolates. Moreover, bin mapping of introgression lines (ILs) derived from the genetically divergent isolates N2 and CB4856 reveals 10 quantitative trait loci (QTLs) affecting dauer formation. Comparison of individual ILs to N2 identifies an additional eight QTLs, and sequential IL analysis reveals six more QTLs. Our results also show that a behavioural, laboratory-derived, mutation controlled by the neuropeptide Y receptor homolog npr-1 can affect dauer larvae development in growing populations. These findings illustrate the complex genetic architecture of variation in dauer larvae formation in C. elegans and may help to understand how the control of variation in dauer larvae development has evolved.     

Hc-daf-2 encodes an insulin-like receptor kinase in the barber’s pole worm, Haemonchus contortus, and restores partial dauer regulation

Infective L3s (iL3s) of parasitic nematodes share common behavioural, morphological and developmental characteristics with the developmentally arrested (dauer) larvae of the free-living nematode Caenorhabditis elegans. It is proposed that similar molecular mechanisms regulate entry into or exit from the dauer stage in C. elegans, and the transition from free-living to parasitic forms of parasitic nematodes. In C. elegans, one of the key factors regulating the dauer transition is the insulin-like receptor (designated Ce-DAF-2) encoded by the gene Ce-daf-2. However, nothing is known about DAF-2 homologues in most parasitic nematodes. Here, using a PCR-based approach, we identified and characterised a gene (Hc-daf-2) and its inferred product (Hc-DAF-2) in Haemonchus contortus (a socioeconomically important parasitic nematode of ruminants). The sequence of Hc-DAF-2 displays significant sequence homology to insulin receptors (IR) in both vertebrates and invertebrates, and contains conserved structural domains. A sequence encoding an important proteolytic motif (RKRR) identified in the predicted peptide sequence of Hc-DAF-2 is consistent with that of the human IR, suggesting that it is involved in the formation of the IR complex. The Hc-daf-2 gene was transcribed in all life stages of H. contortus, with a significant up-regulation in the iL3 compared with other stages. To compare patterns of expression between Hc-daf-2 and Ce-daf-2, reporter constructs fusing the Ce-daf-2 or Hc-daf-2 promoter to sequence encoding GFP were microinjected into the N2 strain of C. elegans, and transgenic lines were established and examined. Both genes showed similar patterns of expression in amphidial (head) neurons, which relate to sensation and signal transduction. Further study by heterologous genetic complementation in a daf-2-deficient strain of C. elegans (CB1370) showed partial rescue of function by Hc-daf-2. Taken together, these findings provide a first insight into the roles of Hc-daf-2/Hc-DAF-2 in the biology and development of H. contortus, particularly in the transition to parasitism.