Targets of TGF-beta signaling in Caenorhabditis elegans dauer formation

Caenorhabditis elegans dauer formation is controlled by multiple environmental factors. The chemosensory neuron ASI regulates dauer formation by secretion of DAF-7/TGF-beta, but the molecular targets of the DAF-7 ligand are incompletely defined and the cellular targets are unknown. We genetically characterized and cloned a putative transducer of DAF-7 signaling called daf-14 and found that it encodes a Smad protein. DAF-14 Smad has a highly unusual structure completely lacking the N-terminal domain found in all other Smad proteins known to date. daf-14 genetically interacts with daf-8, which encodes another Smad, and the interaction suggests partial functional redundancy between these two Smad proteins. We also studied the cellular targets of DAF-7 signaling by studying the sites of action of daf-14 and daf-4, the putative receptor for DAF-7. daf-14::gfp is expressed in multiple tissues that are remodeled during dauer formation. However, analysis of mosaics generated by free duplication loss and tissue-specific expression constructs indicate cell-nonautonomous function of daf-4, arguing against direct DAF-7 signaling to tissues throughout the animal. Instead, these experiments suggest the nervous system as a target of DAF-7 signaling and that the nervous system in turn regulates dauer formation by other tissues.     

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.     

DAF-7/TGF-beta expression required for the normal larval development in C. elegans is controlled by a presumed guanylyl cyclase DAF-11.

In C. elegans development, unfavorable growth conditions lead a larva to an arrested and enduring form called a dauer. To elucidate components upstream of DAF-7/TGF-beta in this control pathway, we isolated a mutant that was defective in daf-7 promoter::gfp reporter expression and showed an arrested (dauer-constitutive) phenotype. It has a new mutation in the daf-11 gene encoding a transmembrane guanylyl cyclase. We show that daf-11 gene and a related gene daf-21 act upstream of daf-7, and cilium-related genes che-2 and che-3 are placed between daf-11 and daf-7, in the genetic pathway controlling dauer formation. Expression of daf-11 cDNA by cell specific promoters suggests that daf-11 acts cell autonomously in ASI chemosensory neurons for daf-7 expression.     

C. elegans DAF-18/PTEN mediates nutrient-dependent arrest of cell cycle and growth in the germline

The molecular pathways that link nutritional cues to developmental programs are poorly understood. Caenorhabditis elegans hatchlings arrest in a dormant state termed "L1 diapause" until food is supplied. However, little is known about what signal transduction pathways mediate nutritional status to control arrest and initiation of postembryonic development. We report that C. elegans embryonic germline precursors undergo G2 arrest with condensed chromosomes and remain arrested throughout L1 diapause. Loss of the DAF-18/PTEN tumor suppressor bypasses this arrest, resulting in inappropriate germline growth dependent on the AGE-1/PI-3 and AKT-1/PKB kinases. DAF-18 also regulates an insulin/IGF-like pathway essential for longevity and dauer larva formation. However, DAF-16/FoxO, which is repressed by this pathway, is not required for germline arrest in L1 diapause. Thus, these findings indicate that quiescence of germline development during L1 diapause is not a passive consequence of nutrient deprivation, but rather is actively maintained by DAF-18 through a pathway distinct from that which regulates longevity and dauer formation.     

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 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.     

Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN

The tumour suppressor gene PTEN (also called MMAC1 or TEP1) is somatically mutated in a variety of cancer types [1] [2] [3] [4]. In addition, germline mutation of PTEN is responsible for two dominantly inherited, related cancer syndromes called Cowden disease and Bannayan-Ruvalcaba-Riley syndrome [4]. PTEN encodes a dual-specificity phosphatase that inhibits cell spreading and migration partly by inhibiting integrin-mediated signalling [5] [6] [7]. Furthermore, PTEN regulates the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by specifically dephosphorylating position 3 on the inositol ring [8]. We report here that the dauer formation gene daf-18 is the Caenorhabditis elegans homologue of PTEN. DAF-18 is a component of the insulin-like signalling pathway controlling entry into diapause and adult longevity that is regulated by the DAF-2 receptor tyrosine kinase and the AGE-1 PI 3-kinase [9]. Others have shown that mutation of daf-18 suppresses the life extension and constitutive dauer formation associated with daf-2 or age-1 mutants. Similarly, we show that inactivation of daf-18 by RNA-mediated interference mimics this suppression, and that a wild-type daf-18 transgene rescues the dauer defect. These results indicate that PTEN/daf-18 antagonizes the DAF-2-AGE-1 pathway, perhaps by catalyzing dephosphorylation of the PIP3 generated by AGE-1. These data further support the notion that mutations of PTEN contribute to the development of human neoplasia through an aberrant activation of the PI 3-kinase signalling cascade.     

SDF-9, a protein tyrosine phosphatase-like molecule,regulates the L3/dauer developmental decision through hormonal signaling in C. elegans

The dauer larva of the nematode Caenorhabditis elegans is a good model system for investigating the regulation of developmental fates by environmental cues. Here we show that SDF-9, a protein tyrosine phosphatase-like molecule, is involved in the regulation of dauer larva formation. The dauer larva of sdf-9 mutants is different from a normal dauer larva but resembles the dauer-like larva of daf-9 and daf-12 dauer-constitutive mutants. Like these mutants, the dauer-constitutive phenotypes of sdf-9 mutants were greatly enhanced by cholesterol deprivation. Epistasis analyses, together with the relationship between sdf-9 mutations and daf-9 expression, suggested that SDF-9 increases the activity of DAF-9 or helps the execution of the DAF-9 function. SDF-9 was expressed in two head cells in which DAF-9 is expressed. By their position and by genetic mosaic experiments, we identified these cells as XXXL/R cells, which are known as embryonic hypodermal cells and whose function at later stages is unknown. Killing of the sdf-9-expressing cells in the wild-type first-stage larva induced formation of the dauer-like larva. Since this study on SDF-9 and former studies on DAF-9 showed that the functions of these proteins are related to those of steroids, XXXL/R cells seem to play a key role in the metabolism or function of a steroid hormone(s) that acts in dauer regulation.     

A Caenorhabditis elegans type I TGF beta receptor can function in the absence of type II kinase to promote larval development

The daf-4 gene encodes a type II bone morphogenetic protein receptor in Caenorhabditis elegans that regulates dauer larva formation, body size and male tail patterning. The putative type I receptor partner for DAF-4 in regulating dauer larva formation is DAF-1. Genetic tests of the mechanism of activation of these receptors show that DAF-1 can signal in the absence of DAF-4 kinase activity. A daf-1 mutation enhances dauer formation in a daf-4 null background, whereas overexpression of daf-1 partially rescues a daf-4 mutant. DAF-1 alone cannot fully compensate for the loss of DAF-4 activity, indicating that nondauer development normally results from the activities of both receptors. DAF-1 signaling in the absence of a type II kinase is unique in the type I receptor family. The activity may be an evolutionary remnant, owing to daf-1's origin near the type I/type II divergence, or it may be an innovation that evolved in nematodes. daf-1 and daf-4 promoters both mediated expression of green fluorescent protein in the nervous system, indicating that a DAF-1/DAF-4 receptor complex may activate a neuronal signaling pathway. Signaling from a strong DAF-1/DAF-4 receptor complex or a weaker DAF-1 receptor alone may provide larvae with more precise control of the dauer/nondauer decision in a range of environmental conditions.     

Dafadine inhibits DAF-9 to promote dauer formation and longevity of Caenorhabditis elegans

The DAF-9 cytochrome P450 is a key regulator of dauer formation, developmental timing and longevity in the nematode Caenorhabditis elegans. Here we describe the first identified chemical inhibitor of DAF-9 and the first reported small-molecule tool that robustly induces dauer formation in typical culture conditions. This molecule (called dafadine) also inhibits the mammalian ortholog of DAF-9(CYP27A1), suggesting that dafadine can be used to interrogate developmental control and longevity in other animals.     

Gαo and Gαq)regulate the expression of daf-7, a TGFβ-like gene, in Caenorhabditis elegans

Caenorhabditis elegans enter an alternate developmental stage called dauer in unfavorable conditions such as starvation, overcrowding, or high temperature. Several evolutionarily conserved signaling pathways control dauer formation. DAF-7/TGFβ and serotonin, important ligands in these signaling pathways, affect not only dauer formation, but also the expression of one another. The heterotrimeric G proteins GOA-1 (Gα(o)) and EGL-30 (Gα(q)) mediate serotonin signaling as well as serotonin biosynthesis in C. elegans. It is not known whether GOA-1 or EGL-30 also affect dauer formation and/or daf-7 expression, which are both modulated in part by serotonin. The purpose of this study is to better understand the relationship between proteins important for neuronal signaling and developmental plasticity in both C. elegans and humans. Using promoter-GFP transgenic worms, it was determined that both goa-1 and egl-30 regulate daf-7 expression during larval development. In addition, the normal daf-7 response to high temperature or starvation was altered in goa-1 and egl-30 mutants. Despite the effect of goa-1 and egl-30 mutations on daf-7 expression in various environmental conditions, there was no effect of the mutations on dauer formation. This paper provides evidence that while goa-1 and egl-30 are important for normal daf-7 expression, mutations in these genes are not sufficient to disrupt dauer formation.     

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.