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C. elegans VANG-1 modulates life span via insulin/IGF-1-like signaling.
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MedLine Citation:
PMID:  22359667     Owner:  NLM     Status:  MEDLINE    
The planar cell polarity (PCP) pathway is highly conserved from Drosophila to humans and a PCP-like pathway has recently been described in the nematode Caenorhabditis elegans. The developmental function of this pathway is to coordinate the orientation of cells or structures within the plane of an epithelium or to organize cell-cell intercalation required for correct morphogenesis. Here, we describe a novel role of VANG-1, the only C. elegans ortholog of the conserved PCP component Strabismus/Van Gogh. We show that two alleles of vang-1 and depletion of the protein by RNAi cause an increase of mean life span up to 40%. Consistent with the longevity phenotype vang-1 animals also show enhanced resistance to thermal- and oxidative stress and decreased lipofuscin accumulation. In addition, vang-1 mutants show defects like reduced brood size, decreased ovulation rate and prolonged reproductive span, which are also related to gerontogenes. The germline, but not the intestine or neurons, seems to be the primary site of vang-1 function. Life span extension in vang-1 mutants depends on the insulin/IGF-1-like receptor DAF-2 and DAF-16/FoxO transcription factor. RNAi against the phase II detoxification transcription factor SKN-1/Nrf2 also reduced vang-1 life span that might be explained by gradual inhibition of insulin/IGF-1-like signaling in vang-1. This is the first time that a key player of the PCP pathway is shown to be involved in the insulin/IGF-1-like signaling dependent modulation of life span in C. elegans.
Sebastian J Honnen; Christian Büchter; Verena Schröder; Michael Hoffmann; Yuji Kohara; Andreas Kampkötter; Olaf Bossinger
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-02-16
Journal Detail:
Title:  PloS one     Volume:  7     ISSN:  1932-6203     ISO Abbreviation:  PLoS ONE     Publication Date:  2012  
Date Detail:
Created Date:  2012-02-23     Completed Date:  2012-08-03     Revised Date:  2013-06-26    
Medline Journal Info:
Nlm Unique ID:  101285081     Medline TA:  PLoS One     Country:  United States    
Other Details:
Languages:  eng     Pagination:  e32183     Citation Subset:  IM    
Institute of Molecular and Cellular Anatomy, Medical School, RWTH Aachen University, Aachen, Germany.
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MeSH Terms
Caenorhabditis elegans / genetics,  metabolism*
Caenorhabditis elegans Proteins / genetics,  physiology*
Cell Polarity
Heat-Shock Response
Insulin / metabolism*
Insulin-Like Growth Factor I / metabolism*
Oxidative Stress
Phosphoproteins / genetics,  physiology*
RNA, Small Interfering / pharmacology
Receptor, Insulin
Signal Transduction*
Transcription Factors
Reg. No./Substance:
0/Caenorhabditis elegans Proteins; 0/DAF-2 protein, C elegans; 0/Insulin; 0/Phosphoproteins; 0/RNA, Small Interfering; 0/Transcription Factors; 0/Vang-1 protein, C elegans; 0/daf-16 protein, C elegans; 67763-96-6/Insulin-Like Growth Factor I; EC, Insulin

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Journal ID (nlm-ta): PLoS One
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
ISSN: 1932-6203
Publisher: Public Library of Science, San Francisco, USA
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Honnen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received Day: 8 Month: 9 Year: 2011
Accepted Day: 23 Month: 1 Year: 2012
collection publication date: Year: 2012
Electronic publication date: Day: 16 Month: 2 Year: 2012
Volume: 7 Issue: 2
E-location ID: e32183
ID: 3281126
PubMed Id: 22359667
Publisher Id: PONE-D-11-17701
DOI: 10.1371/journal.pone.0032183

C. elegans VANG-1 Modulates Life Span via Insulin/IGF-1-Like Signaling Alternate Title:VANG-1 Modulates Life Span
Sebastian J. Honnen12
Christian Büchter2
Verena Schröder2
Michael Hoffmann3
Yuji Kohara4
Andreas Kampkötter25
Olaf Bossinger1*
Anne C. Hartedit1 Role: Editor
1Institute of Molecular and Cellular Anatomy, Medical School, RWTH Aachen University, Aachen, Germany
2Institute of Toxicology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
3Department of General Pediatrics, University Children's Hospital, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
4Genome Biology Laboratory, National Institute of Genetics, Mishima, Japan
5Research and Development, Bayer Animal Health GmbH, Leverkusen, Germany
Brown University, United States of America
Correspondence: * E-mail:
Contributed by footnote: Conceived and designed the experiments: SH AK OB. Performed the experiments: SH CB VS. Analyzed the data: SH OB. Contributed reagents/materials/analysis tools: MH YK. Wrote the paper: SH OB.


Wnt/planar cell polarity (PCP) is one of three identified Wnt signaling pathways, along with Wnt/ß–Catenin and Wnt/Calcium [1]. These signaling pathways are abundant in various developmental processes across the animal kingdom [2][6]. PCP is extensively studied in the Drosophila wing, or in the organization of ommatidia in the fly eye or hair follicles in mammalian skin. Six proteins were placed in the core PCP pathway, Frizzled (Fz), Dishevelled (Dsh), Diego (Dgo), Strabismus/Van Gogh (Stbm/Vang), Prickle (Pk) and Flamingo (Fmi). The signaling mediated by PCP core proteins during development contributes to the polarization alongside the epithelial anterior-posterior or proximo-distal axis and requires contrary clustering of PCP components at the respective cell cortex. As a consequence of PCP signaling, downstream effectors (e.g., the actin cytoskeleton) are polarized within individual cells that finally lead to well organized structures within the two-dimensional epithelial surface [7], [8]. PCP processes also shape three-dimensional tissues that do not exhibit obvious signs of planar polarity. Here, individual cells have to move in a specific direction or divide with a specific orientation, hence showing transient planar polarization (e.g., during mediolateral cell intercalation) required for morphogenesis of the neural tube in vertebrates [9], [10]. Novel components of PCP signaling have been identified in the recent years, and the number of crosslinks to other conserved pathways required for development is rising [11][13].

The C. elegans genome ( encodes a sole four-pass transmembrane protein, VANG-1 showing sequence similarities and conservation of overall domain architecture compared to the Strabismus/Van Gogh/Ltap proteins identified in Drosophila, Xenopus and mammals. Like in Drosophila and mammals, VANG-1 contains four hydrophobic transmembrane domains at its N-terminus and a consensus PDZ binding motif at its C-terminus [13]. VANG-1 was implicated in playing a minor role in B cell polarity in the C. elegans male tail [14]. However, it plays a major role in organ formation either by mediating correct intercalation of intestinal primordial cells during embryogenesis [13], [15] or by establishing ground polarity in vulval development [12]. Whereas PCP signaling required for morphogenesis is generally well understood, a more physiological role of this pathway with effects on metabolism has not been described so far.

C. elegans is a well-established model to study genes that contribute to the process of aging. The corresponding genes of “Age” mutants are referred to as gerontogenes [16]. These mutants share a specific catalog of defects, e.g., a minimum of 20% life span increase and resistance against certain stress factors like reactive oxygen species or heat. The C. elegans homolog of insulin receptor in mammals, daf-2, is one of the best described gerontogenes, and the signaling mediated by DAF-2 is well understood [17][19]. DAF-2 is capable to phosphorylate target substrates, e.g., AGE-1/AAP-1, a PI3 kinase that generates PI(3,4,5)P3 [20][22]. Via a phosphorylation cascade, downstream kinases PDK-1, AKT-1, AKT-2 and SGK-1 [23][25] are activated and in turn negatively regulate the forkhead transcription factor (FoxO), DAF-16 [26], [27]. Inhibition of DAF-2 signaling (e.g., by daf-2 mutations or active insulin peptide signaling) leads to dephosphorylation, activation and accumulation of DAF-16 in the nucleus [28]. Consequently, transcription of DAF-16 targets that include genes involved in defence against stresses, DNA repair and metabolism lead to a higher resistance against stresses and significantly extension of life span [29]. Besides DAF-16, inhibiting insulin/IGF-1-like signaling also activates heat-shock transcription factor HSF-1 and phase II detoxification transcription factor SKN-1, a Nrf1/2/3 protein ortholog [30], [31].

In the present study, we identify VANG-1, the only C. elegans ortholog of the conserved PCP protein Strabismus/Van Gogh, as a gerontogene with a typical phenotype, including extended life- and reproductive span, multiple stress resistances, slow growth, reduced brood size and reduced lipofuscin accumulation. The vang-1–dependent life span extension and stress defences seem to be coordinated in the germline and mostly require daf-16 and skn-1 gene functions.

Results and Discussion
vang-1 increases life span, stress resistance and reproductive span in C. elegans

The C. elegans genome ( contains a sole four-pass transmembrane protein with homology to the Strabismus/Van Gogh/Ltap proteins identified in Drosophila, Xenopus and mammals [32][34]. During analysis of vang-1(tm1422), in which 188 amino acids of the N-terminus are missing (including three of the four transmembrane domains; see supporting information S1) [13], we noticed several defects (Figs. 1, 2) with regard to the postembryonic phenotype, e.g., slow growth (data not shown) and reduced fecundity (Fig. 2A) that are also associated with loss of function phenotypes of certain aging genes in C. elegans[35]. Life span assays in vang-1(tm1422) at 25°C (Fig. 1A), 20°C and 18°C (Table 1) detected a significant increase in mean life span of up to 40% compared to wild type (WT) animals. Furthermore, we tested life span of another vang-1 deletion mutation, ok1142, lacking 162 amino acids of the C-terminus (including a predicted phosphorylation site; see supporting information S1) [13] and of animals depleted of VANG-1 by RNAi (Fig. 1A). Again, we noticed a significant extension of C. elegans mean life span up to 27% and 20% in comparison to WT controls either kept on standard OP50 or RNAi HT115 bacteria with the empty “feeding”-vector. In addition, the tm1422 phenotype was not enhanced by RNAi against vang-1 C-terminus (Table 1). With regard to longevity we assume tm1422 to be a null mutation whereas ok1142 seems to be a hypomorphic allele and RNAi does not generate the complete loss-of-function phenotype, as reported for other genes [36]. Hence, we used tm1422 in all further experiments.

Next, we tested increased resistance of tm1422 against various stressors, a typical feature of gerontogenes. First, we measured thermoresistance in semi-automated and manual assays using SYTOX® Green nucleic acid stain under lethal temperature conditions of 37°C. Both assays revealed increased thermoresistance of tm1422 of about 40% (Fig. 1B and not shown), which is in the similar range as extension of mean life span. Second, we tested the resistance of tm1422 against reactive oxygen species (ROS) and determined intracellular ROS accumulation in living worms. In a stress assay with juglone (from Juglans niger) as a redox cycler, we found the fraction of tm1422 animals that survived the induced ROS stress conditions was about four fold higher than WT (Fig. 1C). Furthermore, a 60% decrease in ROS accumulation, which is in the similar range of daf-2 mutant population (Fig. 1D), was found in tm1422 worms in comparison to WT using a fluorescence well-plate reader to measure DCF fluorescence (see “methods” for details). According to the “free radical theory of aging” [37], ROS are a crucial factor for aging, and the intracellular amount of ROS can be correlated to stress. Organisms developed inducible detoxification systems like catalases, peroxidases and superoxide dismutases to reduce ROS levels [38]. The competence to keep intracellular ROS levels low is considered to be one possibility for the extension of life span [39], [40]. Thus, the diminished amounts of ROS in tm1422 may explain the increased survival rate at lethal thermal stress conditions. Recent findings suggest that the relationship between ROS and the aging process is more complex than what was originally thought. The generation of ROS cannot be longer seen as the initial trigger of the aging process [41]. Nevertheless, in case of tm1422 population reduced ROS generation indicates a lower stress level that finally may account for the extension of life span.

In order to estimate the biological age of vang-1 mutants, we measured the amount of lipofuscin, a product of oxidative damage and autophagy. In C. elegans, lipofuscin is detectable as autofluorescent granules in the intestine and its accumulation is a well-established marker to judge the biological age of C. elegans[42], [43]. In comparison to WT, ok1142 and tm1422 animals showed a significantly decreased accumulation of lipofuscin after five (Fig. 2B) and even after ten days (Fig. 2C).

In addition to the longevity phenotype, we observed a significantly reduced brood size in tm1422 animals (Fig. 2A), a decreased ovulation rate (Fig. 2D) and a dramatically prolonged reproductive span (Fig. 2E). Normally, the reproductive system of C. elegans ages significantly during the first week of adulthood [44], reflected by germline degeneration and a decline in oocyte quality [42], [45]. Individual tm1422 mothers continue to produce viable progeny as they age (Fig. 2D). This phenotype also points to vang-1 being a typical gerontogene. Some of the known mutations (e.g., daf-4 or daf-7) that extend C. elegans' reproductive period also regulate longevity, suggesting that there is a link between reproductive span and life span [46].

Taken together these results suggest that loss of the planar cell polarity ortholog VANG-1 causes robust temperature independent extension of life span, increases stress resistance and extends reproductive period in C. elegans.

Life span modulation by VANG-1 depends on the insulin/IGF-1-like signaling pathway

The main regulator of longevity and stress resistance in C. elegans is insulin/IGF-1-like signaling with its effector DAF-16. This FoxO transcription factor is translocated into the nucleus where it activates gene expression for distinct processes, e.g., resistance against different stressors and longevity when insulin/IGF-1-like signaling is inhibited [47], [48]. To gain further insight into the pathway operating in tm1422, we disrupted FoxO/DAF-16 transcription factor by RNAi in tm1422 and WT worms and compared the mean life span (Fig. 3A and Table 1). As expected [49], mean life span in WT animals depleted of DAF-16 slightly decreased in comparison to the control. Surprisingly, daf-16(RNAi) in tm1422 eliminated vang-1 induced life span extension at 20°C and 25°C (Table 1), suggesting that daf-16 is epistatic to vang-1.

The activation of the DAF-16 transcription factor can be easily observed by a functional DAF-16::GFP fusion [50]. After vang-1(RNAi) at room temperature and 27°C we observed 16% and 57% DAF-16 translocation into the nucleus, respectively (Fig. S1), suggesting that complete nuclear localization of DAF-16 is not a prerequisite for increased life span and stress resistance. This phenomenon has also been observed in case of age-1 at 20°C, which is well known for modulating life span in a DAF-16 dependent manner [48], [50].

To further validate our daf-16(RNAi) life span result, we investigated other parameters of high DAF-16 activity (e.g., developmental arrest). In C. elegans, the activity of DAF-16 is sufficient and necessary for L1 diapause and dauer formation [25], [51]. Hatching L1 larvae stay in diapause, a developmental arrested state with reduced metabolism, until they start feeding. Dauer formation is an alternative third larval stage (beside the normal L3 larval stage) that is introduced under harsh environmental conditions, high temperature, low food or overcrowding [52].

We performed our dauer assay in comparison to WT, daf-2(e1370) and daf-16(mu86) at 27°C [53]. Consistent with the literature, we found that daf-2(e1370), encoding the sole insulin receptor homologue in C. elegans[20], is dauer constitutive (∼99% arrest), while daf-16(mu86) is dauer defective (0% arrest; Fig. 3B) [51], [54]. tm1422 animals showed four times more developmental arrest compared to WT (Fig. 3B), which is inhibited by RNAi against daf-16 (tm1422: 7.8% dauer, 92.2% “other”, n = 64; WT: 1.2% dauer, 98.8% “other”, n = 160;). While 21% of WT animals developed into dauers, 58% and 18% of tm1422 animals arrested as dauers and in L1 diapause, respectively (Fig. 3B). A noteworthy difference concerning the dauer constitutive phenotypes of daf-2 and tm1422 is the percentage of L1 diapause arrests, which is also induced by DAF-16 [51] and suggests higher activity of DAF-16 in tm1422 during early development.

We further investigated the role of the receptor tyrosine kinase DAF-2 [20], which acts upstream of FoxO/DAF-16 transcription factor to modulate life span and stress resistance in the conserved insulin/IGF-1-like signaling pathway [55]. Inhibition or loss of DAF-2 function leads to induction of alternate dauer formation (see above) early in life and life span extension of up to 100% late in life both depending on DAF-16 function [29]. RNAi against daf-2 in WT and tm1422 worms resulted in nearly identical survival curves with no significant difference in mean life span (Fig. 3A and Table 1), indicating that vang-1 may function in the insulin/IGF-1-like signaling pathway, rather than in parallel pathways, e.g., through regulation of DAF-16 by kri-1 and lipophilic-hormone signaling [56], [57].

We also tested the longevity promoting factor SKN-1/Nrf2, which orchestrates the phase II detoxification response including defense against oxidative stress [58]. RNAi against skn-1 did reduce tm1422 life span significantly about 17% (Table 1). Inhibition of insulin/IGF-1-like signaling in tm1422 may explain this result. Like DAF-16, SKN-1 is also repressed by DAF-2 downstream kinases, AKT-1/2 and SGK-1 and possibly acts as a key player in a positive feedback loop to extend life span [58], [59].

To further specify how vang-1 functions in the extension of life span, we performed specific knock downs of vang-1 first in the intestine [60], [61], second in the germline [62] and third, because of its expression in ventral cord neurons [12], [63], in strains showing enhanced neuronal RNAi [64].

The intestine is highly exposed to environmental toxins and pathogens and it has been speculated to be the major site of stress response [65]. To further support this hypothesis, we depleted DAF-2 (as a control) by RNAi only in the intestine and found a 60% extension of life span (Table 1). In contrast, vang-1(RNAi) in the intestine did not result in a significant extension of mean life span (Fig. 4A; Table 1), suggesting that the intestine is not where VANG-1 is acting to modulate life span.

In C. elegans and mice, VANG-1 and Vangl2Lp have been connected with correct uterine epithelium development in the reproductive tract [12], [66], but its function in meiotic maturation and ovulation is still ellusive. Both processes are regulated by intense signaling between the germline and the proximal gonadal sheath cells, specialized myo-epithelial cells that surround and form gap junctions with oocytes [67][70]. During ovulation, sheath cells contract rapidly, the distal constriction of the spermatheca dilates, and sheath cells pull the distal spermatheca over the mature oocyte [71]. The decreased fertility/brood size, ovulation rate, and the increased reproduction span of tm1422 animals (Fig. 2 A,D–E) suggests VANG-1 being involved in the communication between germline and somatic gonad. To test if vang-1 also acts in the germline to control life span by insulin/IGF-1-like signaling, we performed germline-specific RNAi [62]. vang-1(RNAi) in rrf-1 led to a significant increase in life span (13.5%, Fig. 4B; Table 1), which is about two third of whole life span extension observed in vang-1(RNAi) animals (Fig. 1A; Table 1). In contrast, depletion of VANG-1 in the enhanced-neuronal RNAi strains TU3311 and TU3401 [64], has no effect on C. elegans life span extension (Fig. 4C; Table 1). As suggested by Calixto et al. [64] the neuronal expression of sid-1 in TU3311 might serve as a sink for double-stranded RNA used by non-neuronal RNAi and thus could explain why vang-1(RNAi) in TU3311 leads not to the same life span extension as in WT. Additionally, vang-1(tm1422) individuals have an intact chemosensory apparatus and are “open” to the environment (personal comunication with N.J. Storm - it has been testet two times with up to 30 individuals per experiment for uptake of DiI [72]). Dye-fill defective (dyf-phenotype) mutants have previous been found long-lived [73]. Taken together, our findings of tissue-specific RNAi against vang-1 in combination with in-situ hybridization data of vang-1, daf-2 and daf-16 (Fig. S2) implicate the germline to be the primary site of vang-1 action concerning longevity in C. elegans. Components of the insulin/IGF-1-like signaling pathway have already been implicated to act in the germline, e.g., Michaelson et al. found that the effect of reducing daf-2 signaling on larval germline proliferation is dependent on daf-16[74].

In summary, we have identified a link between the C. elegans planar cell polarity key player vang-1 and insulin/IGF-1-dependent extension of life span. Mutations in vang-1 show the typical phenotype of age-mutants, including longevity, slow growth, multiple stress resistances, reduced lipofuscin accumulation, and reduced brood size. The germline, but not the intestine or neurons seems to be the primary site of vang-1 function, which may operate in the same pathway as daf-2 and daf-16 to extend life span of about 40% in C. elegans.

C. elegans strains and alleles

Maintenance and handling of C. elegans were carried out as described previously [75]. Bristol N2 was used as the WT strain. WT or mutant worms were synchronized as described previously [76].

Single mutants were as follows

TM1422: vang-1(tm1422)X (outcrossed ×3); RB1125: vang-1(ok1142) X; CB1370: daf-2(e1370) III; CF1038: daf-16(mu86) I; NL2098: rrf-1(pk1417) I.

Transgenic strains were as follows

OLB11: rde-1(ne219);[pOLB11(elt-2p::rde-1)+pRF4(rol-6(su1006))]; TU3311: [unc-119p::YFP+unc-119p::sid-1]; TU3401: sid-1(pk3321) V; [pCFJ90(myo-2p::mCherry)+unc-119p::sid-1]; TJ356: integrated DAF-16::GFP roller strain [50] (for further details see:

RNA-mediated interference (RNAi)

RNAi by “feeding” was performed essentially as described by others [77]. In brief, after amplification of a single colony overnight (37°C, LBamp tet medium), HT115(DE3) bacteria (RNase III-deficient E. coli strain, carrying IPTG-inducible T7-polymerase) [77], [78] were diluted to an OD600 of 0.9, and after addition of IPTG (1 mM) seeded on NGMamp tet plates (containing 1 mM IPTG). Bacteria were further incubated overnight at room temperature (∼22°C) to allow the expression of double-stranded RNA. HT115(DE3) bacteria harboring the empty KS+ based vector L4440 (containing two T7 promoters flanking a polylinker) were used as a control for RNAi “feeding” experiments. RNAi clones against vang-1 and daf-16 were obtained from the Ahringer RNAi “feeding”-library (Geneservice Limited, Cambridge, UK) while daf-2 “feeding”-clone was kindly provided by Dr. Andrew Dillin [79] (see supporting information S2 for sequencing results of RNAi “feeding”-clones).

Life span assay

Life span was determined at 25°C, if not stated otherwise. Because vang-1(tm1422) shows a delayed egg laying phenotype, synchronization was performed as follows: embryos were randomly collected from cut-off worms, transferred and grown on plates (three plates per trial) either seeded with OP50 [75] or HT115(DE3) [77], [78] bacteria harboring the empty L4440 “feeding”-vector or L4440 with a fragment of the gene of interest [77]. Worms were transferred to fresh plates every day during time of reproduction but at least every third day. Individuals were considered as dead when stopped moving and not responded to gentle touches. When dying upon “rupture”/“bag of worms” phenotypes or disappearance occurred, the animal was censored on that day. The resulting data sets were analyzed using Kaplan-Meier survival test and weighted log-rank tests [80].

Determination of progeny

C. elegans populations were synchronized and hatched on NGM Agar plates at 25°C. On day three, single worms were transferred as L4 larvae to 35 mm NGM-plates with NGM agar. Adult worms were transferred to fresh plates and then their progenies were counted each day. The experiment was stopped when production of progeny ceased.

Dauer assay

The assay was performed as described elsewhere [53]. In brief, some gravid adults were put on individual tagged 60 mm NGM-plates where they laid eggs for 4–6 h at 20°C before they were removed again. Plates were shifted to the assay temperature of 27°C. After 60 h the stages were scored for L1 diapause and dauers. Farther grown worms (individuals larger than L2 larvae but not predauer/dauer stages) were pooled as “other”.

Reproductive span of self-fertile animals (modified after [46])

Ten hermaphrodites per trial were individually transferred to fresh 35 mm NGM-plates seeded with OP50 daily. No production of progeny for 48 h marked reproductive cessation. Individuals were censored if they died or matricide occurred. All trials were conducted at 20°C with age synchronized populations. Unpaired t-test was used to test null hypothesis.

Determination of ovulation rate (modified after [71])

Documentation of ovulation rates were performed using a Zeiss Axioplan 2 microscope. Age-synchronized worms with more than six oocytes in-utero were transferred to small agarose pads on a microscope slide and coated with a cover slip. The number of ovulated oocytes per animal was counted for 3 h and slides were kept in a moisture chamber at room temperature.

DAF-16::GFP translocation

Synchronized populations of TJ356 (DAF-16::GFP) [50] worms were kept for 72 h at 25°C on NGM plates seeded with RNAi HT115 bacteria either carrying the empty “feeding”-vector or a fragment of vang-1 cDNA. 15 Individuals per trial were transferred to small agarose pads (3%) on a microscope slide, anesthetized with levamisole (1%), coated with a cover slip, illuminated with UV light under an Axiolab fluorescence microscope (Zeiss, Göttingen, Germany) and dedicated to three categories concerning DAF-16::GFP translocation: “cytoplasmatic” (uniform distribution of DAF-16::GFP), “intermediate” (clearly distinguishable DAF-16::GFP in some nuclei), and “nuclear” (DAF-16::GFP in nearly all nuclei with low background fluorescence).

Lipofuscin accumulation

WT C. elegans were synchronized, hatched on NGM Agar plates at 20°C and transferred to fresh plates every second day. At day five and day ten, individuals were placed on microscope slides, anaesthetized with 20 mM sodium azide in M9 buffer [76] and coated with a cover slip. Epifluorescence (excitation, 365 nm; emission, 420 nm) images were taken with Image ProPlus software (Version 4.5, MediaCybernetics, Silver Spring, MD, USA) using a CoolSnap CF Digital Monochrome Camera (Intas, Göttingen, Germany) mounted on an Axiolab fluorescence microscope (Zeiss, Göttingen, Germany) and using a 100× oil immersion objective. The fluorescence intensity of individual worms was determined densitometrically as relative fluorescence units (RFU: ODindividual−ODbackground/mm2).

Assessment of resistance to thermal/oxidative stress and determination of intracellular ROS accumulation in C. elegans

The resistance of WT and mutant animals to thermal stress was assessed by a semi-automated assay according to [81] with some modifications described in [82]. After synchronization [76] both strains were cultured on NGM plates with OP50 bacteria [75] for five days at 20°C. Worms were then washed in PBST (PBS/0.1% Tween 20) and individually transferred with 1 µl PBST to the wells of a 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany, #788096) containing 9 µl PBST with 1×107 OP50 bacteria/ml [82]. Immediately after transfer 10 µl of 2 µM SYTOX® Green nucleic acid stain (Molecular Probes Inc., Leiden, Netherlands) in PBS was added to the wells and the plate was sealed using BackSeal-96/384 Black (Perkin Elmer, Wellesley, USA, #6005189) to avoid evaporation. SYTOX® Green can only enter cells with compromised plasma membranes and exerts a bright fluorescence in the DNA-bound state. Therefore, the fluorescence intensity is an indicator for cellular damage and hence for the viability of worms [81]. For the application of thermal stress the fluorescence reader (Wallace Victor2 1420 multilabel counter, Perkin Elmer, Wellesley, USA) was preheated to 37°C. The measurement of each well through the transparent bottom of the microtiter plate (excitation, 485 nm; emission, 535 nm) was carried out for a minimum of 13 h with intervals of 15 min and a 0.2 s integration time. Fluorescence curves for every single well were obtained and individual cut off values were determined by multiplying the background fluorescence (average of the first four measurement readings) by a factor of three [81][83]. The time point when fluorescence exceeded the cut off value was defined as the point of death of the corresponding worm and the survival curves as well as the mean life spans were assessed from these individual times of death.

To compare resistance to oxidative stress WT and mutant animals synchronized [76] and L4 larvae were incubated for approximate 5 h at 20°C in liquid NGM containing 200 µM juglone, a redox cycler that generates intracellular oxidative stress [84]. Worms were then allowed to regenerate on NGM plates with OP50 bacteria [75] for about 20 h at 20°C before viability was determined by touch provoked movement [85].

For the determination of the intracellular amount of ROS synchronized WT and mutant larvae were cultured as described above and individually transferred with 1 µl PBST to the wells of a 384-well microtiter plate containing 7 µl PBS [86]. After the complete transfer of the individual worms 2 µl 250 µM 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA; Molecular Probes Inc., Leiden, Netherlands) in PBS (final concentration, 50 µM) was added to the wells and the plate was sealed (see above). After entering cells H2DCF-DA is intracellular converted to membrane-impermeable, non-fluorescent H2DCF, which then can be oxidized by ROS to yield fluorescent DCF and thus is a marker for the individual amount of intracellular ROS in a single worm [82], [83], [86]. The fluorescence of each well is then measured through the transparent bottom in a fluorescence reader (see above) every 15 min for a minimum of 13 h at 37°C (1.0 s integration time; excitation, 485 nm; emission, 535 nm).

Supporting Information Figure S1

DAF-16::GFP translocation into the nucleus. In TJ356 (DAF-16::GFP) worms [50], RNAi against vang-1 at room temperature (RT) led to 12% and 4% intermediate and nuclear localization of DAF-16::GFP, respectively (n = 49*). In contrast, TJ356 control animals fed with RNAi HT115 bacteria, carrying the empty “feeding”-vector, showed 100% cytoplasmic localization of DAF-16::GFP (n = 70*). Under heat stress condition (27°C), vang-1(RNAi) causes 45% and 12% intermediate and nuclear localization of DAF-16::GFP, respectively (n = 42*). In comparison, TJ356 control animals showed 42% intermediate- and 3% nuclear localization of DAF-16::GFP (n = 95*). *(p<0.05 by two-way ANOVA with Bonferroni's post hoc test; three or more independent trials).


Click here for additional data file (pone.0032183.s001.png)

Figure S2

Expression patterns in C. elegans adults of daf-2 (A), daf-16 (B) and vang-1 (C) genes. All images represent in situ hybridization to endogenous transcripts (enriched in the gonad, arrows) and are taken from the Nematode Expression Data Base ( Scale bars: 60 µm.


Click here for additional data file (pone.0032183.s002.png)

Supporting Information S1

Sequences of VANG-1, VANG-1tm1422 and VANG-1ok1142 proteins. Missing amino acids in tm1422 and ok1142 are shown in red and blue, respectively. Additional amino acids in ok1142 are shown in yellow. For further details concerning VANG-1 see [13].


Click here for additional data file (pone.0032183.s003.docx)

Supporting Information S2

Sequences of RNAi “feeding”-clones.


Click here for additional data file (pone.0032183.s004.docx)


Competing Interests: Andreas Kampkötter is employed by Bayer Animal Health GmbH. However, this does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Funding: Deutsche Forschungsgemeinschaft (SFB590 TP B3) to OB. SH is a fellow of the Jürgen Manchot Stiftung. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors would like to thank: C. Cowan, A. Wodarz, A. Müller and J. Nelson for critical reading of the manuscript, N.J. Storm for sharing data, A. Dillin for providing daf-2 RNAi “feeding”-clone, the Mitani lab and the C. elegans Knockout Consortium for providing tm1422 and ok1142, respectively. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR).

1. Nelson WJ,Nusse R. Year: 2004Convergence of Wnt, beta-catenin, and cadherin pathways.Science3031483148715001769
2. Cadigan K,Nusse R. Year: 1997Wnt signaling: a common theme in animal development.Genes And Development11328633059407023
3. Wodarz A,Nusse R. Year: 1998Mechanisms of Wnt signaling in development.Annu Rev Cell Dev Biol1459889891778
4. Wang Y,Nathans J. Year: 2007Tissue/planar cell polarity in vertebrates: new insights and new questions.Development13464765817259302
5. Wu J,Mlodzik M. Year: 2009A quest for the mechanism regulating global planar cell polarity of tissues.Trends Cell Biol1929530519560358
6. Axelrod JD. Year: 2009Progress and challenges in understanding planar cell polarity signaling.Semin Cell Dev Biol2096497119665570
7. Goodrich LV,Strutt D. Year: 2011Principles of planar polarity in animal development.Development1381877189221521735
8. Jenny A. Year: 2010Planar cell polarity signaling in the Drosophila eye.Current topics in developmental biology9318922720959167
9. Keller R. Year: 2002Shaping the vertebrate body plan by polarized embryonic cell movements.Science2981950195412471247
10. Keller R,Shook D. Year: 2008Dynamic determinations: patterning the cell behaviours that close the amphibian blastopore.Philos Trans R Soc Lond B Biol Sci3631317133218192174
11. Mirkovic I,Gault WJ,Rahnama M,Jenny A,Gaengel K,et al. Year: 2011Nemo kinase phosphorylates beta-catenin to promote ommatidial rotation and connects core PCP factors to E-cadherin-beta-catenin.Nature structural & molecular biology
12. Green J,Inoue T,Sternberg P. Year: 2008Opposing Wnt pathways orient cell polarity during organogenesis.Cell13464665618724937
13. Hoffmann M,Segbert C,Helbig G,Bossinger O. Year: 2010Intestinal tube formation in Caenorhabditis elegans requires vang-1 and egl-15 signaling.Dev Biol33926827920004187
14. Wu M,Herman MA. Year: 2006A novel noncanonical Wnt pathway is involved in the regulation of the asymmetric B cell division in C. elegans.Dev Biol29331632916631156
15. Leung B,Hermann GJ,Priess JR. Year: 1999Organogenesis of the Caenorhabditis elegans intestine.Dev Biol21611413410588867
16. Johnson TE,Henderson S,Murakami S,de Castro E,de Castro SH,et al. Year: 2002Longevity genes in the nematode Caenorhabditis elegans also mediate increased resistance to stress and prevent disease.J Inherit Metab Dis2519720612137228
17. Kenyon CJ. Year: 2010The genetics of ageing.Nature46450451220336132
18. Antebi A. Year: 2007Genetics of aging in Caenorhabditis elegans.PLoS Genet31565157117907808
19. Wolff S,Dillin A. Year: 2006The trifecta of aging in Caenorhabditis elegans.Exp Gerontol4189490316919905
20. Friedman D,Johnson T. Year: 1988A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility.Genetics11875868608934
21. Morris J,Tissenbaum H,Ruvkun G. Year: 1996A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans.Nature3825365398700226
22. Wolkow CA,Munoz MJ,Riddle DL,Ruvkun G. Year: 2002Insulin receptor substrate and p55 orthologous adaptor proteins function in the Caenorhabditis elegans daf-2/insulin-like signaling pathway.J Biol Chem277495914959712393910
23. Hertweck M,Göbel C,Baumeister R. Year: 2004C. elegans SGK-1 is the critical component in the Akt/PKB kinase complex to control stress response and life span.Dev Cell657758815068796
24. Paradis S,Ailion M,Toker AS,Thomas JH,Ruvkun G. Year: 1999A PDK1 homolog is necessary and sufficient to transduce AGE-1 PI3 kinase signals that regulate diapause in Caenorhabditis elegans.Genes Dev131438145210364160
25. Paradis S,Ruvkun G. Year: 1998Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor.Genes Dev12248824989716402
26. Ogg S,Paradis S,Gottlieb S,Patterson GI,Lee L,et al. Year: 1997The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans.Nature3899949999353126
27. Lin KT,Dorman J,Rodan A,Kenyon C. Year: 1997daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegansScience278131913229360933
28. Huang H,Tindall DJ. Year: 2007Dynamic FoxO transcription factors.J Cell Sci1202479248717646672
29. Kenyon C,Chang JW,Gensch E,Rudner A,Tabtiang R. Year: 1993A C. elegans mutant that lives twice as long as wild type.Nature3664614648247153
30. An JH,Blackwell TK. Year: 2003SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response.Genes & development171882189312869585
31. Hsu AL,Murphy CT,Kenyon C. Year: 2003Regulation of aging and age-related disease by DAF-16 and heat-shock factor.Science3001142114512750521
32. Darken RS,Scola AM,Rakeman AS,Das G,Mlodzik M,et al. Year: 2002The planar polarity gene strabismus regulates convergent extension movements in Xenopus.Embo J2197698511867525
33. Goto T,Keller R. Year: 2002The planar cell polarity gene strabismus regulates convergence and extension and neural fold closure in Xenopus.Dev Biol24716518112074560
34. Kibar Z,Vogan KJ,Groulx N,Justice MJ,Underhill DA,et al. Year: 2001Ltap, a mammalian homolog of Drosophila Strabismus/Van Gogh, is altered in the mouse neural tube mutant Loop-tail.Nat Genet2825125511431695
35. Luscombe NM,Qian J,Zhang Z,Johnson T,Gerstein M. Year: 2002The dominance of the population by a selected few: power-law behaviour applies to a wide variety of genomic properties.Genome Biol3RESEARCH004012186647
36. Guo S,Kemphues KJ. Year: 1996A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans.Nature3824554588684486
37. Harman D. Year: 1956Aging: a theory based on free radical and radiation chemistry.J Gerontol1129830013332224
38. Braeckman B,Houthoofd K,Vanfleteren J. Year: 2002Assessing metabolic activity in aging Caenorhabditis elegans: concepts and controversies.Aging Cell18288; discussion 102–10312882336
39. Finkel T,Holbrook NJ. Year: 2000Oxidants, oxidative stress and the biology of ageing.Nature40823924711089981
40. Van Raamsdonk JM,Hekimi S. Year: 2010Reactive Oxygen Species and Aging in Caenorhabditis elegans: Causal or Casual Relationship?Antioxid Redox Signal131911195320568954
41. Hekimi S,Lapointe J,Wen Y. Year: 2011Taking a “good” look at free radicals in the aging process.Trends in Cell Biology
42. Garigan D,Hsu AL,Fraser AG,Kamath RS,Ahringer J,et al. Year: 2002Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation.Genetics1611101111212136014
43. Gerstbrein B,Stamatas G,Kollias N,Driscoll M. Year: 2005In vivo spectrofluorimetry reveals endogenous biomarkers that report healthspan and dietary restriction in Caenorhabditis elegans.Aging Cell412713715924569
44. Hughes SC,Evason K,Xiong C,Kornfeld K. Year: 2007Genetic and pharmacological factors that influence reproductive aging in nematodes.PLoS Genet3e2517305431
45. Andux S,Ellis RE. Year: 2008Apoptosis maintains oocyte quality in aging Caenorhabditis elegans females.PLoS Genet4e100029519057674
46. Luo S,Shaw WM,Ashraf J,Murphy CT. Year: 2009TGF-beta Sma/Mab signaling mutations uncouple reproductive aging from somatic aging.PLoS Genet5e100078920041217
47. Kenyon C. Year: 2005The plasticity of aging: insights from long-lived mutants.Cell12044946015734678
48. Lin KT,Hsin H,Libina N,Kenyon C. Year: 2001Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling.Nat Genet2813914511381260
49. Samuelson AV,Carr CE,Ruvkun G. Year: 2007Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants.Genes Dev212976299418006689
50. Henderson S,Johnson TE. Year: 2001daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans.Curr Biol111975198011747825
51. Baugh LR,Sternberg PW. Year: 2006DAF-16/FOXO regulates transcription of cki-1/Cip/Kip and repression of lin-4 during C. elegans L1 arrest.Curr Biol1678078516631585
52. Gerisch B,Weitzel C,Kober-Eisermann C,Rottiers V,Antebi A. Year: 2001A hormonal signaling pathway influencing C. elegans metabolism, reproductive development, and life span.Dev Cell184185111740945
53. Hu PJ,Xu J,Ruvkun G. Year: 2006Two membrane-associated tyrosine phosphatase homologs potentiate C. elegans AKT-1/PKB signaling.PLoS Genet2e9916839187
54. Gottlieb S,Ruvkun G. Year: 1994daf-2, daf-16 and daf-23: genetically interacting genes controlling Dauer formation in Caenorhabditis elegans.Genetics1371071208056303
55. Landis JN,Murphy CT. Year: 2010Integration of diverse inputs in the regulation of Caenorhabditis elegans DAF-16/FOXO.Dev Dyn
56. Berman JR,Kenyon C. Year: 2006Germ-cell loss extends C. elegans life span through regulation of DAF-16 by kri-1 and lipophilic-hormone signaling.Cell1241055106816530050
57. Hsin H,Kenyon C. Year: 1999Signals from the reproductive system regulate the lifespan of C. elegans.Nature39936236610360574
58. Tullet JM,Hertweck M,An JH,Baker J,Hwang JY,et al. Year: 2008Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans.Cell1321025103818358814
59. Okuyama T,Inoue H,Ookuma S,Satoh T,Kano K,et al. Year: 2010The ERK MAPK pathway regulates longevity through SKN-1 and insulin-like signaling in C. elegans.J Biol Chem
60. Pilipiuk J,Lefebvre C,Wiesenfahrt T,Legouis R,Bossinger O. Year: 2009Increased IP3/Ca2+ signaling compensates depletion of LET-413/DLG-1 in C. elegans epithelial junction assembly.Dev Biol327344719109941
61. McGhee JD,Fukushige T,Krause MW,Minnema SE,Goszczynski B,et al. Year: 2009ELT-2 is the predominant transcription factor controlling differentiation and function of the C. elegans intestine, from embryo to adult.Dev Biol32755156519111532
62. Sijen T,Fleenor J,Simmer F,Thijssen KL,Parrish S,et al. Year: 2001On the role of RNA amplification in dsRNA-triggered gene silencing.Cell10746547611719187
63. Sanchez-Alvarez L,Visanuvimol J,McEwan A,Su A,Imai JH,et al. Year: 2011VANG-1 and PRKL-1 Cooperate to Negatively Regulate Neurite Formation in Caenorhabditis elegans.PLoS genetics7e100225721912529
64. Calixto A,Chelur D,Topalidou I,Chen X,Chalfie M. Year: 2010Enhanced neuronal RNAi in C. elegans using SID-1.Nature methods755455920512143
65. Libina N,Berman JR,Kenyon C. Year: 2003Tissue-specific activities of C. elegans DAF-16 in the regulation of lifespan.Cell11548950214622602
66. vandenBerg AL,Sassoon DA. Year: 2009Non-canonical Wnt signaling regulates cell polarity in female reproductive tract development via van gogh-like 2.Development1361559157019363157
67. Greenstein D,Hird S,Plasterk RH,Andachi Y,Kohara Y,et al. Year: 1994Targeted mutations in the Caenorhabditis elegans POU homeo box gene ceh-18 cause defects in oocyte cell cycle arrest, gonad migration, and epidermal differentiation.Genes Dev8193519487958868
68. McCarter J,Bartlett B,Dang T,Schedl T. Year: 1997Soma-germ cell interactions in Caenorhabditis elegans: multiple events of hermaphrodite germline development require the somatic sheath and spermathecal lineages.Dev Biol1811211439013925
69. Miller MA,Ruest PJ,Kosinski M,Hanks SK,Greenstein D. Year: 2003An Eph receptor sperm-sensing control mechanism for oocyte meiotic maturation in Caenorhabditis elegans.Genes Dev1718720012533508
70. Rose K,Winfrey V,Hoffman L,Hall D,Furuta T,et al. Year: 1997The POU gene ceh-18 promotes gonadal sheath cell differentiation and function required for meiotic maturation and ovulation in Caenorhabditis elegans.Dev Biol19259779405097
71. McCarter J,Bartlett B,Dang T,Schedl T. Year: 1999On the control of oocyte meiotic maturation and ovulation in Caenorhabditis elegans.Dev Biol2051111289882501
72. Mukhopadhyay A,Deplancke B,Walhout AJ,Tissenbaum HA. Year: 2005C. elegans tubby regulates life span and fat storage by two independent mechanisms.Cell Metab2354216054097
73. Apfeld J,Kenyon C. Year: 1999Regulation of lifespan by sensory perception in Caenorhabditis elegans.Nature40280480910617200
74. Michaelson D,Korta DZ,Capua Y,Hubbard EJ. Year: 2010Insulin signaling promotes germline proliferation in C. elegans.Development13767168020110332
75. Brenner S. Year: 1974The genetics of Caenorhabditis elegans.Genetics7771944366476
76. Lewis JA,Fleming JT. Year: 1995Basic culture methods.Methods Cell Biol483298531730
77. Kamath RS,Martinez-Campos M,Zipperlen P,Fraser AG,Ahringer J. Year: 2001Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.Genome Biol2research 0002.00010002.0010
78. Timmons L,Court DL,Fire A. Year: 2001Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.Gene26310311211223248
79. Cohen E,Bieschke J,Perciavalle RM,Kelly JW,Dillin A. Year: 2006Opposing activities protect against age-onset proteotoxicity.Science3131604161016902091
80. Woolson RF,Clarke WR. Year: 2002Statistical Methods for the Analysis of Biomedical DataNew YorkWiley
81. Gill MS,Olsen A,Sampayo JN,Lithgow GJ. Year: 2003An automated high-throughput assay for survival of the nematode Caenorhabditis elegans.Free Radic Biol Med3555856512957648
82. Kampkötter A,Nkwonkam CG,Zurawski RF,Timpel C,Chovolou Y,et al. Year: 2007Investigations of protective effects of the flavonoids quercetin and rutin on stress resistance in the model organism Caenorhabditis elegans.Toxicology23411312317376580
83. Kampkötter A,Pielarski T,Rohrig R,Timpel C,Chovolou Y,et al. Year: 2007The Ginkgo biloba extract EGb761 reduces stress sensitivity, ROS accumulation and expression of catalase and glutathione S-transferase 4 in Caenorhabditis elegans.Pharmacol Res5513914717207635
84. de Castro E,Hegi de Castro S,Johnson TE. Year: 2004Isolation of long-lived mutants in Caenorhabditis elegans using selection for resistance to juglone.Free Radic Biol Med3713914515203185
85. Lithgow GJ,White TM,Melov S,Johnson TE. Year: 1995Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress.Proc Natl Acad Sci USA92754075447638227
86. Kampkötter A,Gombitang Nkwonkam C,Zurawski RF,Timpel C,Chovolou Y,et al. Year: 2007Effects of the flavonoids kaempferol and fisetin on thermotolerance, oxidative stress and FoxO transcription factor DAF-16 in the model organism Caenorhabditis elegans.Arch Toxicol8184985817551714


[Figure ID: pone-0032183-g001]
doi: 10.1371/journal.pone.0032183.g001.
Figure 1  vang-1 function interferes with life span extension and resistance against high temperature and reactive oxygen species in C. elegans.

(A) vang-1 function interferes with life span extension in C. elegans.tm1422 (red), ok1142 (green) and vang-1(RNAi) (orange) animals showed a significantly extended mean life span (14.3±0.4 d, n = 174*; 12.9±0.5 d, n = 114*; and 14.9±0.2 d, n = 576*, respectively) in comparison to controls: WT animals either grown on OP50 bacteria (light blue; 10.2±0.2 d, n = 214*, p<0.0001**) or RNAi HT115 bacteria (blue; 12.8±0.1 d, n = 936*, p<0.001**). (B–D) vang-1(tm1422) increases resistance to thermal/oxidative stress in C. elegans. (B) At 37°C, the mean survival time of tm1422 (red, 6.2±0.3 h, n = 48*) was significantly increased (p<0.01**) in comparison to WT (blue, 4.3±0.1 h, n = 48*). (C) After 5–6 h under oxidative stress (induced by 200 µM juglone), a significantly larger fraction of tm1422 animals survived (p<0,05***) (red, 34.4±6%, n>100*) in comparison to WT (blue, 5.7±2%, n>100*). (D) After 4 h at 37°C, tm1422 animals (red, 20,020±2,148, n = 48*) and e1370 animals (green, 29,243±2,528, n = 52*) showed a significantly lower DCF (2,7-dichlorofluorescein) fluorescence (p<0.001***) in comparison to WT (blue, 54,911±3,940, n = 48*). (*three or more independent trials, **Mantel-Cox log rank test, ***unpaired t-test; animals grown on OP50 bacteria, if not stated otherwise; results are shown as mean±SEM).

[Figure ID: pone-0032183-g002]
doi: 10.1371/journal.pone.0032183.g002.
Figure 2  vang-1 shows reproduction- and aging-related defects.

(A) vang-1(tm1422) populations have a reduced brood size. The average brood size at 25°C in vang-1(tm1422) (red, 111±41 progeny; n = 28*) is significantly reduced (p<0.0001**) in comparison to WT (blue, 194±50 progeny; n = 56*). Results are shown as mean±standard deviation. (B–C) ok1142 and tm1422 show decreased lipofuscin accumulation five and ten days after hatching. (B) Five days after hatching, ok1142 (green, RFU = 792.35±25, n = 31, p<0.001**) and tm1422 (red, RFU = 543.1±18, n = 37, p<0.001**) accumulate significantly less lipofuscin in comparison to WT (blue, RFU = 900.4±17.27, n = 45). (C) Ten days after hatching, ok1142 (green, RFU = 1083±32, n = 33, p<0.05**) and tm1422 (red, RFU = 940.9±27, n = 29, p<0.01**) still accumulate significantly less lipofuscin in comparison to WT (blue, RFU = 1196±37, n = 27). Results are shown as mean±SEM of relative fluorescence units (RFU: ODindividual−ODbackground/mm2). (D) In tm1422 the ovulation rate is reduced in comparison to WT.tm1422 has an ovulation rate of 0.7±0.1 (n = 25***) and the WT shows 2.3±0.7 (n = 15***) what is significantly more (p<0.05**). Ovulations were counted per gonad arm per hour at 20°C for synchronous WT and mutant populations. (E) vang-1 populations have a prolonged reproductive span. The reproductive span in ok1142 (green, 6.6 d; n = 20***) and tm1422 (red, 6.9 d; n = 20***) is significantly prolonged (p<0.05##) in comparison to WT (blue, 5.7 d; n = 20***). (*three independent trials, **unpaired t-test, ***two independent trials; animals grown on OP50 bacteria, ##Mantel-Cox log rank test).

[Figure ID: pone-0032183-g003]
doi: 10.1371/journal.pone.0032183.g003.
Figure 3  vang-1(tm1422) life span modulation depends on Insulin/IGF-1-like signaling and leads to higher DAF-16 activity.

(A) vang-1(tm1422) induced life span extension interferes with RNAi against daf-2 and daf-16. Depletion of DAF-2 by RNAi in tm1422 (brown spotted line) and WT (green solid line) causes an increase of mean life span to 25.6±1.3 d (n = 135*) and 25.8±1.1 d (n = 126*), respectively (p<0.62**), which is in agreement with published results for daf-2 mutants [23]. In contrast, depletion of DAF-16 by RNAi in tm1422 (rose spotted line) and WT (purple solid line) causes a decrease of mean life span to 12±0.3 d (n = 247*) and 11.9±0.2 d (n = 142*), respectively (p<0.96**). Life spans of WT (blue solid line) and tm1422 (red spotted line) fed with RNAi HT115 bacteria carrying the empty “feeding”-vector are 12.8±0.1 d (n = 936*) and 15.8±0.2 d (n = 480*), respectively (p<0.001**). (B) vang-1(tm1422) populations are dauer constitutive. Synchronous populations were scored after 60 h at 27°C (OP50 bacteria) for dauers and L1 in diapause. All farther grown and adult animals were pooled as “other”. WT animals developed 21%, 0% and 79% dauers, L1 diapause and “other”, respectively (n = 390*). daf-2(e1370) animals showed 94.7%, 4.6% and 0.7% dauers, L1 diapause and “other”, respectively (n = 281*). daf-16(mu86) animals developed 100% “other” (n = 111*). tm1422 showed 57.6%, 18% and 24.4% dauers, L1 diapause and “other”, respectively (n = 205*, p<0.05§). (*three or more independent trials, **Mantel-Cox log rank test, animals grown on OP50 bacteria, if not stated otherwise, §Data analyzed by Chi-square test).

[Figure ID: pone-0032183-g004]
doi: 10.1371/journal.pone.0032183.g004.
Figure 4  Tissue specific RNAi against vang-1.

(A) vang-1 function interferes with life span extension in C. elegans.vang-1(RNAi) (orange) animals showed a significantly extended mean life span (14.9±0.2 d, n = 576*) in comparison to control: RNAi HT115 bacteria (blue; 12.8±0.1 d, n = 936*, p<0.001**). (B) Germline-specific RNAi against vang-1 effects C. elegans life span. After vang-1(RNAi) in germline-specific RNAi strain NL2098 a significant increase (13%) of mean life span (14.6±0.3 d, red spotted line, n = 274*) in comparison to the control (NL2098 kept on RNAi HT115 bacteria carrying the empty “feeding”-vector) can be observed (12.9±0.3 d, blue spotted line, n = 309*, p<0.01**). (C–D) Neuron-specific RNAi against vang-1 does not effect C. elegans life span. After depletion of VANG-1 in the enhanced-neuronal RNAi strain TU3311 ([unc-119p::YFP+unc-119p::sid-1]), the mean life span is 21.1±0.4 d (orange solid line, n = 280*) compared to 19.5±0.5 d (green solid line, n = 92*, p = 0.02**) in the control (TU3311 kept on RNAi HT115 bacteria carrying the empty “feeding”-vector). The same is true in the neuron-specific RNAi strain TU3401 (sid-1(pk3321) V; [pCFJ90(myo-2p::mCherry)+unc-119p::sid-1]), which only has SID-1 in neurons. Depletion of VANG-1 in this strain leads to a mean life span of 17.7±0.3 d (red spotted line, n = 284*) and 17±0.3 d (blue spotted line, n = 380*, no significant difference**) in the control (TU3401 kept on RNAi HT115 bacteria carrying the empty “feeding”-vector). (E) Intestine-specific RNAi against vang-1 does not effect C. elegans life span. After depletion of VANG-1 in the intestine-specific RNAi strain OLB11 {rde-1(ne219);[pOLB11(elt-2p::rde-1)+pRF4(rol-6(su1006))]}, the mean life span is 14.4±0.3 d (red solid line, n = 195*) compared to 14.0±0.3 d (blue solid line, n = 250*, no significant difference**) in the control (OLB11 kept on RNAi HT115 bacteria carrying the empty “feeding”-vector). (*three or more independent trials, **Mantel-Cox log rank test).

[TableWrap ID: pone-0032183-t001] doi: 10.1371/journal.pone.0032183.t001.
Table 1  Summary of life spans.
Background Conditions LS +/− SEM N Significance
1 WT OP50 10.2+/−0.2 214
2 tm1422 OP50 14.3+/−0.4 174 *(1)
3 ok1142 OP50 12.9+/−0.5 114 *(1)
4 WT 18°C/OP50 19.6+/−0.8 61
5 tm1422 18°C/OP50 27.1+/−0.9 43 *(4)
6 WT 20°C/HT115 21.4+/−0.4 70
7 tm1422 20°C/HT115 25.6+/−0.4 71 *(25)
8 WT 20°C/daf-16(RNAi) 19.5+/−0.5 75 *(25)
9 tm1422 20°C/daf-16(RNAi) 22.5+/−0.5 71 *(26) 0.72(25)
10 WT HT115 12.8+/−0.1 936 *(1)
11 tm1422 HT115 15.8+/−0.2 480 *(10)
12 WT vang-1(RNAi) 14.9+/−0.2 576 *(10)
13 tm1422 vang-1(RNAi) 14.9+/−0.4 242 0.37(11)
14 WT skn-1(RNAi) 13.0+/−0.2 133 0.76(10)
15 tm1422 skn-1(RNAi) 13.2+/−0.5 138 *(11)
16 WT daf-16(RNAi) 11.9+/−0.2 142 *(10)/0.27(17)
17 tm1422 daf-16(RNAi) 12+/−0.3 247 *(11)
18 WT daf-2(RNAi) 25.8+/−1.1 126 *(10)/0.62(19)
19 tm1422 daf-2(RNAi) 25.6+/−1.3 135 *(11)
20 OLB11 HT115 14+/−0.3 250 *(10)
21 OLB11 daf-2(RNAi) 22.6+/−0.7 85 *(20)
22 OLB11 vang-1(RNAi) 14.4+/−0.3 195 0.24(20)
23 NL2098(rrf-1) HT115 12.9+/−0.3 309
24 NL2098(rrf-1) vang-1(RNAi) 14.6+/−0.3 274 *(23)
25 TU3401 20°C/HT115 17.0+/−0.3 380
26 TU3401 20°C/vang-1(RNAi) 17.7+/−0.3 284 0.86(25)
27 TU3311 20°C/HT115 19.5+/−0.5 212
28 TU3311 20°C/vang-1(RNAi) 21.1+/−0.4 280 *(27)

Life spans (LS±SEM, standard error of the mean, at 25°C, if not stated otherwise) under different experimental conditions in WT, two different alleles of vang-1 (tm1422 and ok1142), the intestine-specific RNAi strain OLB11 [60], [61], germline-specific RNAi strain NL2098 [62] and the neuron-enhanced and neuron-specific strains TU3311 and TU3401 [64]. OP50 [75] and RNAi HT115 [77], [78] indicate standard and RNAi E. coli strains, respectively. Comparison of significant results are indicated by *(p<0.01; Mantel-Cox log rank test) with corresponding experiments in parentheses (the p-value is stated, if not significant). All the life span assays were repeated at least three times. Data shown is a sum of all experiments.

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