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Dorsal Dauers and Directionally Dysfunctional dex-1 Dumpies

Caroline D. Beshers1,2, Kristen M. Flatt1,3 and Nathan E. Schroeder1,3
1 Crop Sciences Department, University of Illinois Urbana-Champaign
2 Neuroscience Department, Oberlin College, Ohio
3 Neuroscience Program, University of Illinois Urbana-Champaign

Correspondence to: Caroline Beshers (caroline.beshers13@gmail.com)
The dauer life stage in wild-type C. elegans exhibits morphological and behavioral characteristics that differentiate it from non-dauers (Hu, 2007). In addition to the commonly observed radial shrinkage, quiescence and lack of pharyngeal pumping, we noted a tendency of wild-type dauers to lie dorsoventrally when mounted on agarose slides, as opposed to laterally like adults. To quantify this observation, we mounted wild-type dauers and L3s on increasing concentrations of agarose with 0.1 M levamisole. We found that 75-90% of wild-type dauers mounted with levamisole lie dorsoventrally regardless of agarose concentration (Fig. 1). Very few (2.5-5%) non-dauer animals were positioned dorsoventrally. There was no significant difference in positioning between dauers that still retained their L2d cuticle and those that were unsheathed.  We then utilized a non-anesthetic method of immobilization by mounting dauers on 10% agarose with 10 micron polystyrene microbeads (Kim et al., 2013). Interestingly, only 15% of wild type dauers lie dorsoventrally when mounted using the microbeads. We hypothesized that the radial shrinkage and the presence of lateral alae in wild-type dauer cuticles may cause anesthetized dauers to roll to a dorsoventral position, but that “conscious” dauers immobilized with microbeads would remain lateral, despite the presence of alae. To test this, we examined dex-1 mutant dauers. We recently found that dex-1(ns42) dauers are defective for dauer alae formation and radial constriction leading to a dumpy dauer phenotype. We found that dex-1 dauers are significantly more likely to lay laterally than wild-type dauers (p=0.0013) (Fig. 1) suggesting that dauer alae or overall body dimensions regulate the anesthetized “sleeping” position.

Figures



Figure 1: This chart illustrates the difference in percentage of N2 dauers, N2 L3s and dex-1 dauers lying dorsoventrally across slide methods with increasing concentrations of agarose using levamisole (lev) and 10% agarose using polystyrene microbeads (beads). The N2 dauers consistently (75-90%) lay dorsoventrally, while 2-5% of non dauer animals lay dorsoventrally. N2 dauers lay dorsoventrally significantly more than dex-1 dauers (p=.0013).


Figure 2:

References

Hu, P.J. Dauer (August 08, 2007), WormBook, ed. The C. elegans Research Community, WormBook  PubMed

Kim E, Sun L, Gabel CV, Fang-Yen C (2013) Long-Term Imaging of Caenorhabditis elegans Using Nanoparticle-Mediated Immobilization. PLoS ONE 8(1): e53419.   PubMed

The daf-2(e1370) allele is not temperature sensitive, however, the dauer phenotype is temperature sensitive

Collin Yvès Ewald1,2 and T. Keith Blackwell2
1 Eidgenössische Technische Hochschule (ETH) Zürich, Health Sciences and Technology, Schwerzenbach-Zürich, Switzerland
2 Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, United States
Correspondence to: T. Keith Blackwell (keith.blackwell@joslin.harvard.edu)
The daf-2 reference allele e1370 is used widely to assess the function of reduced Insulin/IGF-1 receptor signalling in development, metabolism, stress-defence, and aging. The daf-2(e1370) mutants develop into dauers at higher temperatures (25°C) but not at lower temperatures (15°C; (Gems et al. 1998)). Based on this observation, the assumption is that daf-2(e1370) mutants have lower insulin/IGF-1 signalling (IIS) at 25°C and presumably normal or wild-type IIS at 15°C. However, two pieces of evidence argue against this assumption.

  1. At lower temperature (15°C), the daf-2(e1370) mutants are long-lived to the same extent as at higher temperatures (Figure 1). In addition, at lower temperature (15°C) daf-2(e1370) mutants are also stress resistant (Gems et al. 1998).
  2. The transcription factors DAF-16 and SKN-1 that are phosphorylated directly by IIS, and thereby prohibited from entering the nucleus, are nuclear in daf-2(e1370) mutants at 15°C to the same extent as at higher temperatures (Lin et al. 2001 PMID:11381260; Tullet et al. 2008; Ewald et al. 2015).

These findings suggest that IIS is reduced comparably at 15°C and 25°C and argues against the assumption that e1370 is necessarily a temperature-sensitive allele in the classic sense where temperature affects protein folding and activity. Interestingly, about 10% wild-type worms form dauers when grown at 27°C under otherwise standard culturing conditions (ample food and not overcrowded; (Ailion & Thomas 2000 PMID:11063684)). Furthermore, it has been observed that null mutants in other abnormal-DAuer-Formation (daf) genes than daf-2, only show the dauer phenotype at higher temperatures, suggesting that the dauer phenotype itself is temperature sensitive. This observation was published over 30 years ago (Golden & Riddle 1984) and is discussed in (Wood 1988).

We are motivated to write to the WBG community by the assumption that during adulthood daf-2(e1370) mutants improperly reactive phenotypes that are reminiscent of dauer larvae (reduced mobility, reduced brood-size, short body size; (Gems et al. 1998)). Recently, we have shown that daf-2(e1370) mutants form dauers independent of SKN-1/NRF1,2,3 and that SKN-1 is only required for the daf-2(e1370)-longevity phenotype when these dauer-related phenotypes are not activated during adulthood (Ewald et al. 2015). Furthermore, comparing expression profiles of daf-2(e1370) mutants with dauer larvae revealed that they share only a common overlap under conditions that reactivate these dauer phenotypes (e.g., daf-2(e1370) at higher but not at lower temperatures; (Ruzanov et al. 2007 PMID:17543485; McElwee et al. 2004 PMID:15308663; Ewald et al. 2015)). Finally, treating wild-type worms with dauer pheromone was sufficient to increase lifespan (Kawano et al. 2005 PMID:16377915; Ewald et al. 2015). This suggests that there might be a dauer program activated in daf-2(e1370) mutants at higher temperatures that masks other underlying mechanisms of reduced IIS on lifespan. Further research is required, however, these findings suggest that reduced IIS can extend lifespan via dauer-dependent and dauer-independent mechanisms and this changes the interpretation of experiments performed with daf-2(e1370) mutants under conditions that promote these dauer-associated phenotypes.

Figures



Figure 1: daf-2(e1370) mutants are long-lived at lower temperature

References

Ewald, C.Y. et al., 2015. Dauer-independent insulin/IGF-1-signalling implicates collagen remodelling in longevity. Nature, 519(7541), pp.97–101.  PubMed

Gems, D. et al., 1998. Two pleiotropic classes of daf-2 mutation affect larval arrest, adult behavior, reproduction and longevity in Caenorhabditis elegans. Genetics, 150(1), pp.129–155.  PubMed

Golden, J.W. & Riddle, D.L., 1984. A pheromone-induced developmental switch in Caenorhabditis elegans: Temperature-sensitive mutants reveal a wild-type temperature-dependent process. Proceedings of the National Academy of Sciences of the United States of America, 81(3), pp.819–823.  PubMed

Tullet, J.M.A. et al., 2008. Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans. Cell, 132(6), pp.1025–1038.
  PubMed

Wood, W.B. ed., 1988. The Nematode Caenorhabditis elegans. 1st ed., Cold Spring Harbor Monograph Series.  

L1 arrest affects dauer decision in C. elegans

Alexander B. Artyukhin1,2 and Leon Avery1
1Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond VA, 2Boyce Thompson Institute for Plant Research, Cornell University, Ithaca NY

Correspondence to: Alexander B. Artyukhin (alex_artyukhin@yahoo.com)

During larval development C. elegans has two distinct time points where it can arrest development until environmental conditions become more favorable. The first one is L1 arrest, which occurs when worms hatch in the absence of food. The second decision point is later, around L1-L2 molt, and if conditions are poor at this time (little food, crowding, and high temperature) worms gain the potential to become dauers. L1 arrest and dauer formation are not mutually exclusive and, in principle, a worm can go through both of them sequentially. However, it is not known whether L1 arrest and dauer formation are coupled, i.e., if experiencing L1 arrest affects a worm’s decision to become a dauer. A naïve prediction would be that worms that experienced L1 starvation should be more susceptible to become dauers since they were already exposed to a harsh environment.

We studied dauer formation of N2 worms in liquid and found that in reality worms that experienced L1 arrest and starvation were less likely to become dauers. In a typical experiment, eggs obtained from an egg prep were incubated at 20 0C in S-complete with a small amount of bacterial food (HB101) and dauer formation was assessed 6 days later based on survival in 1% SDS (Pungaliya et al., 2009). When we let eggs hatch without food and added it 1-4 days later, keeping other conditions the same, significantly fewer worms became dauers (Figure 1). This was not due to slower development after starvation and incomplete dauer formation. We also found that this effect reflected an internal state of the worm rather than exposure to L1 exometabolome during L1 arrest since worms starved as L1s at low and high density later formed dauers with the same probability (Figure 1, during the dauer formation worms were at the same low density of 1 worm/µl to minimize the effect of released chemicals, see below).

Starved L1 worms release numerous metabolites, including several ascarosides, and we anticipated that they might promote dauer formation. Indeed, when S-complete was substituted with conditioned medium from high-density L1 larvae starved for 24 h, more worms became dauers (Fig. 2), consistent with the known dauer-inducing effect of crowding mediated by ascarosides (Ludewig and Schroeder, 2013). However, this result apparently contradicts the starvation effect described above. On one hand, when a worm itself experienced L1 arrest but received no signal from other worms (low density condition), it was less likely to become a dauer and preferred to try to grow to adult. On the other hand, a worm that had not experienced starvation itself but was exposed to chemical signals released by other starved L1 worms, was more likely to become a dauer. The next obvious experiment is to combine these two opposite effects and see which one is stronger. The result of this test (Fig. 2) is that the external starvation signal (mediated by L1 conditioned medium) overrides the internal one (L1 arrest). When making a decision whether to become a dauer, apparently C. elegans worms trust the communal voice more than their own.

While the dauer-inducing effect of L1-conditioned medium is intuitively easy to accept, the negative effect of L1 starvation on subsequent dauer formation is less clear. A recently published model of contrast effects (McNamara et al., 2013) may provide a theoretical framework for understanding this phenomenon. But to keep us from being too comfortable with our simplistic views nature presented us with another puzzle: L1 arrest does not negatively affect dauer formation in C. briggsae.

Figures



Figure 1: Percentage of dauers decreased when N2 worms experienced L1 arrest for 2 days. The outcome did not depend on whether worms were L1 arrested at low density (LD, 1 worm/µl) or high density (HD, 36.5 worms/µl). Washing L1 arrested worms before adding bacterial food did not make a difference either.


Figure 2: Exposure to conditioned medium from starved N2 L1s increased percentage of dauers in both non-starved and L1-starved N2 worms. Results for two independent preparations of L1 medium are shown (L1 medium 1: 21.5 h conditioning with 125 worms/µl at 22 oC; L1 medium 2: 22 h conditioning with 72.5 worms/µl at 22 oC). L1 starvation inhibited dauer formation in S-complete control sample but had no significant effect in L1 medium samples.

References

Ludewig AH and Schroeder FC. (2013). Ascaroside signaling in C. elegans (January 18, 2013), WormBook, ed. The C. elegans Research Community. PubMed

McNamara JM, Fawcett TW, and Houston AI. (2013). An adaptive response to uncertainty generates positive and negative contrast effects. Science 340, 1084-1086. PubMed

Pungaliya C, Srinivasan J, Fox BW, Malik RU, Ludewig AH, Sternberg PW, and Schroeder FC. (2009). A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. Proc. Natl Acad. Sci. U. S. A. 106, 7708-7713. PubMed

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