Insulin/Insulin-like growth factor signaling controls non-Dauer developmental speed in the nematode Caenorhabditis elegans

Identified as a major pathway controlling entry in the facultative dauer diapause stage, the DAF-2/Insulin receptor (InsR) signaling acts in multiple developmental and physiological regulation events in Caenorhabditis elegans. Here we identified a role of the insulin-like pathway in controlling developmental speed during the C. elegans second larval stage. This role relies on the canonical DAF-16/FOXO-dependent branch of the insulin-like signaling and is largely independent of dauer formation. Our studies provide further evidence for broad conservation of insulin/insulin-like growth factor (IGF) functions in developmental speed control.     

The second messenger cyclic GMP mediates activation in Ancylostoma caninum infective larvae

The developmentally arrested infective larva (L(3)) of hookworms encounters a host-specific signal during infection that initiates previously suspended developmental pathways. Activated L(3) express a parasitic gene set that encodes proteins involved in moulting, growth and development to the adult stage. Early events in this activation to parasitism can be investigated using an in vitro larval feeding assay. When Ancylostoma caninum L(3) are exposed to a host-like stimulus, they resume feeding and release molecules involved in infection. The dauer larva of the free-living nematode Caenorhabditis elegans is a developmentally arrested stage analogous to the hookworm L(3). Recovery from the dauer stage has been proposed as a model for the transition to parasitism in hookworm. Dauer formation and recovery involve several tightly regulated pathways, including a cyclic GMP mediated signalling pathway. To determine if hookworm L(3) activation uses a similar pathway, 8-bromo-cyclic GMP, a membrane permeant analogue of cyclic GMP, was tested for its ability to stimulate feeding. Populations of L(3) incubated with 0.5 mM 8-bromo-cyclic GMP began feeding, and reached maximum feeding at 3.5-5.0 mM. Unlike the serum stimulus, which triggers feeding after a short exposure, 8-bromo-cyclic GMP must be present throughout the entire incubation. Both serum stimulated and 8-bromo-cyclic GMP stimulated L(3) secreted Ancylostoma secreted protein 1, indicating that the stimuli activate the same pathway. Serum and 8-bromo-cyclic GMP stimulated feeding was inhibited by atropine, a muscarinic receptor antagonist. However, only serum stimulated feeding was inhibited by 4,7-phenanthroline, a non-chelating isomer of the metalloprotease inhibitor 1,10-phenantholine. The results indicate that cyclic GMP mediates activation in hookworm larvae, and that a muscarinic receptor is involved in activation. This also suggests that hookworm activation and dauer recovery share similar signalling pathways, and that C. elegans dauer recovery can be used as a model for the transition to parasitism in hookworms.     

Inhibition of germline proliferation during C. elegans dauer development requires PTEN, LKB1 and AMPK signalling

In C. elegans, reduced insulin-like signalling induces developmental quiescence, reproductive delay and lifespan extension. We show here that the C. elegans orthologues of LKB1 and AMPK cooperate during conditions of reduced insulin-like signalling to establish cell cycle quiescence in the germline stem cell population, in addition to prolonging lifespan. The inactivation of either protein causes aberrant germline proliferation during diapause-like ;dauer' development, whereas the loss of AMPK uncouples developmental arrest from lifespan extension. Reduced TGF-beta activity also triggers developmental quiescence independent of the insulin-like pathway. Our data suggest that these two signalling pathways converge on the C. elegans PTEN orthologue to coordinate germline proliferation with somatic development during dauer formation, via the regulation of AMPK and its upstream activator LKB1, rather than through the canonical insulin-like signalling cascade. In humans, germline mutations in TGF-beta family members, PTEN or LKB1 result in related tumour-predisposing syndromes. Our findings establish a developmental relationship that may underscore their shared, characteristic aetiology.     

Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.     

Transforming growth factor-beta and insulin-like signalling pathways in parasitic helminths

The signal transduction pathways involved in regulating developmental arrest in the free-living nematode, Caenorhabditis elegans, are fairly well characterised. However, much less is known about how these processes may influence the developmental timing and maturation in helminth parasites. Here, we provide an overview of two signalling pathways implicated in the regulation of dauer larva formation in C. elegans, the insulin-like signalling pathway and the transforming growth factor-beta pathway, and explore what is known about these signalling pathways in a variety of parasitic helminths. Understanding the differences about how these pathways are affected by environmental cues in free-living versus parasitic species of helminths may provide insights into novel mechanisms for the control or prevention of helminth-induced disease.     

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.     

Phosphoinositide-3-OH-kinase inhibitor LY294002 prevents activation of Ancylostoma caninum and Ancylostoma ceylanicum third-stage infective larvae

The developmentally arrested hookworm infective larva resumes development only after encountering specific host-mediated cues during invasion. These cues activate a signaling pathway that culminates in the resumption of development. In Ancylostoma caninum, activation is characterised by the resumption of feeding and the release of excretory/secretory products required for infection. The dauer stage of the free-living nematode Caenorhabditis elegans is a developmentally arrested stage analogous to the hookworm infective larva. Dauer larvae exit developmental arrest in response to environmental cues that indicate favorable conditions for reproduction and growth. Because of the similarity between dauer recovery and activation, exit from dauer provides a model for hookworm larval activation. An insulin-signaling pathway has been implicated in controlling exit from developmental arrest in both C. elegans dauers and A. caninum larvae. To further investigate the role of insulin signaling in hookworm larval activation, the phosphatidylinositol-3-OH kinase inhibitor LY294002 was tested for its effect on in vitro activation using the resumption of feeding as a marker for activation. LY294002 prevented feeding in A. caninum infective larvae stimulated with host serum filtrate and a glutathione-analogue, the muscarinic agonist arecoline, or the cell permeable cGMP-analogue 8-bromo-cGMP. Similar results were seen with the congeneric hookworm Ancylostoma ceylanicum. These data suggest that an insulin-signaling pathway mediates activation in hookworm larvae, as in C. elegans, and that the phosphatidylinositol-3-OH kinase inhibitor acts downstream of the cGMP and muscarinic signaling steps in the pathway. In A. caninum, LY294002 had no effect on the release of excretory/secretory products associated with activation, suggesting that the secretory pathway diverges from the activation pathway upstream of the phosphatidylinositol-3-OH kinase step. These results provide additional support for the insulin-signaling pathway as the primarily pathway for activation to parasitism in hookworm larvae.     

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. Here, we show that a 34 nucleotide region of the glp-1 3' UTR contains two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, we show that the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor, and contributes to the spatial organization of Notch signaling.     

Eukaryotic translation initiation factor 5B activity regulates larval growth rate and germline development in Caenorhabditis elegans

In C. elegans, a population of proliferating germ cells is maintained via GLP-1/Notch signaling; in the absence of GLP-1 signaling, germ cells prematurely enter meiosis and differentiate. We previously identified ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we report that iffb-1 (initiation factor five B) is an ego gene. iffb-1 encodes the sole C. elegans isoform of eukaryotic translation initiation factor 5B, a protein essential for translation. We have used RNA interference and a deletion mutation to determine the developmental consequences of reduced iffb-1 activity. Our data indicate that maternal iffb-1 gene expression is sufficient for embryogenesis, and zygotic iffb-1 expression is required for development beyond late L1/early L2 stage. Partial reduction in iffb-1 expression delays larval development and can severely disrupt proliferation and differentiation of germ cells. We hypothesize that germline development is particularly sensitive to iffb-1 expression level.     

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.     

Insulin receptor substrate and p55 orthologous adaptor proteins function in the Caenorhabditis elegans daf-2/insulin-like signaling pathway

An insulin-like signaling pathway regulates development and lifespan in Caenorhabditis elegans. Genetic screens that identified many components of the C. elegans insulin pathway did not identify homologs of insulin receptor substrates or the phosphoinositide 3-kinase (PI3K) adaptor/regulatory subunit, which are both required for signaling by mammalian insulin/insulin-like growth factor I pathways. The C. elegans genome contains one homolog of each protein. The C. elegans versions of insulin receptor substrate (IST-1) and PI3K p50/p55 (AAP-1) share moderate sequence similarity with their vertebrate and Drosophila counterparts. Genetic experiments show that ist-1 and aap-1 potentiate C. elegans insulin-like signaling, although they are not required for signaling in the pathway under most conditions. Worms lacking AAP-1 activity because of the mutation aap-1(m889) constitutively arrest development at the dauer larval stage when raised at high temperatures. aap-1 mutants also live longer than wild-type animals, a phenotype observed in other C. elegans mutants with defects in DAF-2 signaling. Interestingly, IST-1 appears to be required for signaling through a pathway that may act in parallel to AGE-1/PI3K.     

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.     

Chemosensory control of surface antigen switching in the nematode Caenorhabditis elegans

Nematodes change their surface compositions in response to environmental signals, which may allow them to survive attacks from microbial pathogens or host immune systems. In the free-living species Caenorhabditis elegans, wild-type worms are induced to display an L1 (first larval stage) surface epitope at later larval stages when grown on an extract of spent culture medium (Inducible Larval Display or ILD). Before this study, it was not known whether ILD was regulated by the well-characterized, neurologically based chemical senses of C. elegans, which mediate other behavioural and developmental responses to environmental signals such as chemotaxis and formation of the facultatively arrested dauer larva stage. We show here that ILD requires the activities of three genes that are essential for the function of the C. elegans chemosensory neurons. ILD was abolished in chemotaxis-defective che-3, osm-3 and tax-4 mutants. In contrast, chemotaxis-defective mutants altered in a different gene, srf-6, show constitutive display of the L1 epitope on all four larval stages. The ILD-defective che-3, osm-3 and tax-4 mutations blocked the constitutive larval display of an srf-6 mutant. Combining srf-6 and certain dauer-constitutive mutations in double mutants enhanced constitutive dauer formation, consistent with the idea that srf-6 acts in parallel with specific components of the dauer formation pathway. These results taken together are consistent with the hypothesis that ILD is triggered by environmental signals detected by the nematode's chemosensory neurons.