Insulin-Like Signaling and Tissue-Specific Requirements of Anoxia Survival in Caenorhabditis elegans


Oxygen deprivation (anoxia or hypoxia) is central to the pathology of various medical problems (heart attack, stroke, solid tumor cancer cells) leading to severe economic and human health consequences. Many organisms can survive oxygen deprivation, and it is of interest to study the mechanisms they employ to do this. The soil nematode Caenorhabditis elegans can survive one day of anoxia, and mutations in the daf-2/daf-16 pathway can increase this length of time (Padilla, Nystul, Zager, Johnson, & Roth, 2002; Mendenhall, LaRue, & Padilla, 2006). I investigated daf-2mutants in anoxia and found that mutations in the ligand binding site of Class 1 mutants allowed C. elegans to survive long term anoxia at very high rates. Additionally, I was interested in seeing if expression of the DAF-16 transcription factor in certain tissues would increase C. elegans anoxia survival as it was shown to increase lifespan when expressed in the intestine (Libina, Berman, & Kenyon, 2004). Unlike longevity, expression of DAF-16 only increased anoxia survival when expressed in the neurons as compared with DAF-16 in the muscle or intestine. These findings provide an understanding for the development of anoxia-related therapies in humans, and genetic treatments of human diseases.

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    The purpose of this project was to gain a greater understanding of how the daf-2/daf-16 insulin-like genetic pathway in the nematode C. elegans contributes to the animal’s anoxia survival. This includes understanding which specific daf-2 mutations are necessary for C. elegans’ anoxia survival, as well as which tissue with DAF-16 expression facilitates greatest anoxia survival.

    Rationale for Research

    The most common cause of death in developed countries is tissue damage from ischemic necrosis during heart attacks and strokes (Griselli, et al., 1999). Ischemic necrosis occurs when an insufficient amount of oxygen reaches the cell, creating an anoxic environment, and leading to stroke. Harm from such diseases has been linked to damage done to tissues after oxygen is circulated back into the system. A number of therapeutic strategies have been devised to minimize such damage (Griselli, et al., 1999). The C-reactive protein (CRP), which is synthesized by hepatocytes, has been implicated in causing damage to tissues. While the reasons for the presence of the CRP are not clearly understood, it is known that during inflammation, infection, etc., the amount of protein synthesized rises by a factor of several thousand, and greatly increases the damage to tissues (Kitsis & Jialal, 2006). A therapeutic strategy that has been effective in decreasing CRP related tissue damage in mice involves the small molecule inhibitor 1, 6-bis(phosphocholine)-hexane (Pepys, et al., 2006). I was interested in exploring alternative, genetic approaches to solve this problem. I wanted to identify the genes responsible for facilitating oxygen recirculation without the concomitant tissue damage. This would presumably lead to an approach that is inherently more efficient than protein inhibition because altering the genes involved would attack the problem in a more holistic and permanent manner compared with protein inhibition.

    However, studying the effects of a particular gene is almost impossible because of the complex molecular mechanisms at work in the human body. An appropriate model system can circumvent this hurdle by giving scientists a similar biological system with which to work. Some characteristics that would make an organism a good model system include short generation time, sequenced genome, and conserved metabolic and developmental pathways. The soil nematode C. elegans has all these features, which has made it useful in the study of many human diseases such as diabetes, Parkinson’s disease and heavy-metal contamination (Forsythe, et al., 2006).

    Of particular interest to me was the daf-2/daf-16 insulin-like signaling pathway which has been implicated in conferring stress resistance in C. elegans. This pathway is homologous to the human FoxO pathway, which is important for basic cell processes such as metabolism, aging, and protecting the body against cancer (Paik, et al., 2007). I wanted to determine if certain daf-2 mutants would survive anoxia better than others, and from these data deduce a mechanism by which the genes and their byproducts influence anoxia survival. Additionally, I was interested in determining if a specific tissue known to express the DAF-16 transcription factor under stress would give the worm an advantage in anoxia survival. I hypothesized that certain daf-2 mutants would survive anoxia longer than non-mutated (wild-type) worms, and also that certain tissues with DAF-16 expression would have a disproportionate impact on anoxia survival. Uncovering the details of how the daf-2/daf-16 pathway works in C. elegans to allow it to cope with environmental stress would give researchers a greater understanding of the human FoxO pathway. Knowing which molecules C. elegans synthesizes to help survive long-term anoxia would be especially important in developing therapies for patients recovering from strokes and heart attacks.


    The daf-2 pathway is primarily involved in initiating the dauer response in C. elegans. The dauer larvae is the third stage larvae that forms when the worm experiences adverse environmental conditions, such as starvation and desiccation (Golden & Riddle, 1984). In this stage, the worm shrinks in diameter, does not feed, and is also resistant to paraquat treatment, heat shock, and other stresses. When favorable conditions return, the worm reenters the regular life cycle and continues development until it reaches adulthood (Gottlieb & Ruvkun, 1994).

    By inactivating certain genes in this pathway, scientists can correlate stress responses with particular genes. Previous research on the nematode showed that a single mutation in the daf-2 pathway, the daf-2(e1370), doubles the lifespan of the nematode (Kenyon, Chang, Gensch, Rudner, & Tabtiang, 1993). Subsequent research led to the classification of daf-2 mutants into two classes based primarily on stress resistance, and in particular thermo-tolerance, longevity, and dauer constituency (Gems, et al., 1998). Other scientists showed that expression of the DAF-16 transcription factor in intestinal tissues of daf-2 mutants increased the nematode’s lifespan (Libina, Berman, & Kenyon, 2004). This and other evidence firmly established that the daf-2/daf-16 pathway is responsible for stress resistance and longevity. The homology of the DAF-16 transcription factor to the human FOXO transcription factor makes it especially significant to scientists. This process is illustrated in Figure 1.

    Initially, members of the Padilla research group uncovered differences in the anoxia survival of different developmental stages of the wild-type C. elegans (Padilla, Nystul, Zager, Johnson, & Roth, 2002). Embryos and larvae could survive anoxia for a full day with viabilities of 90% or higher. By contrast, the dauer larvae (the stage that the nematode enters during stress) could survive anoxia for several days, making this developmental state most resistant to anoxia. Further experiments showed that the daf-2(e1370) mutant could survive long-term anoxia (longer than 3 days of <0.001 kPa O2, 20 ºC), and high temperature anoxia (one day of <0.001 kPa O2, 28 ºC). Additionally, two other genes, gpd-2 and gpd-3, which encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were shown to play an important role in anoxia survival, both long-term and high temperature (Mendenhall, LaRue, & Padilla, 2006). Based on these findings, it seemed possible that other daf-2 mutations might also influence anoxia survival, and that the DAF-16 transcription factor expressed under tissue specific promoters would influence anoxia survival differently. In order to investigate these possibilities, I devised a multi-pronged investigation designed to answer the following research questions (RQ):

    RQ1: Do Class 1 genetic mutations increase nematode anoxia survival compared to non-mutated animals? For how long?

    • daf-2(e1368)
    • daf-2(m577)
    • daf-2(m41)

    RQ2: Do Class 2 genetic mutations increase nematode anoxia survival compared to non-mutated animals? For how long?

    • daf-2(e979)
    • daf-2(e1391)

    RQ3: Is there a difference between Class 1 and Class 2 mutations in the length ofnematode anoxia survival?

    RQ4: Does DAF-16 expression in different tissues (intestinal, neuronal, and muscle) affect nematode anoxia survival differently?


    Strains and Growth Conditions

    The wild-type Bristol strain (N2) was used as a control for all experiments. All mutant strains were ordered from the Caenorhabditis Genetics Center (CGC). All worms, wild-type and mutants strains were cultured using nematode growth media (NGM) plates seeded with Escherichia coli (OP50) and raised at 20 ºC as described (Sulston & Hodgkin, 1988). Synchronized populations of animals were obtained from hypochlorite-treated adults. The embryos were then allowed to hatch, and grow to a young adult stage, which is ~24 hours after the L4 stage. Due to differences in developmental times for daf-2 mutants, as opposed to the wild-type control, daf-2 worms were hypochlorite-treated a full day before the wild-type, to allow for young adults to be synchronized.

    Oxygen-deprivation experiments

    For all experiments, the nematodes were exposed to an anoxic environment using the BioBag Type A environment chamber (Padilla, et al., 2002). The anoxia exposure time (3 or 5 days) and the temperature conditions (20 ºC) were kept constant, and are noted in each experiment.

    Anoxia Survival Assays

    Nematodes at the young adult stage were used for each experiment. For each assay, two plates were placed into the BioBag: one plate containing 50 daf-2 mutants and the other plate containing a similar number of wild-type animals. After the length of the experiment (3 or 5 days) the bags were cut open, and plates taken out and placed at 20ºC at normoxic conditions. This gave the nematodes time to exit the suspended state and made it easier to assess which ones were alive. Worms were considered alive if they were moving, or if they moved after being prodded with a platinum wire. If they did not move after being prodded five times, they were scored as dead.


    I was interested in understanding if certain mutations in the daf-2 pathway would lead to anoxia survival of C. elegans. Subsequent trials of both classes of alleles were carried out in 3 and 5 days of anoxia. Table 1 presents data for the Class 2 alleles which I tested for 3 day anoxia survival: daf-2(1391) and daf-2(e979). This is subsequently graphed in Figure 2. I noticed no survivors after 5 days of anoxia (results not shown). Class 1 mutants all survived 3 days of anoxia at high viabilities (results not shown), so I decided to test their survival at 5 days. Table 2 represents the survival percentages of Class 1 mutants after 5 days of anoxia. Unlike the Class 2 mutations, two Class 1 mutants daf-2(e1368) and daf-2(m41) had high viabilities even after 5 days of anoxia (Figure 3).

    Expression of certain genes can be transgenically regulated, for example by placing the daf-16 gene under the unc-119 promoter, the DAF-16 transcription factor would be only expressed in neuronal tissues. Previous studies had shown that the DAF-16 transcription factor expressed under the intestinal promoter ges-1 can increase the lifespan of C. elegans, and I was interested in testing these strains in anoxia to understand which tissues with DAF-16 would be especially important. I tested transgenic strains that had the transcription factor DAF-16 expressed in neurons (unc-119), muscle (myo-3), and intestine (ges-1) tissues. These mutants were tested at 3 days of anoxia with wild-type controls and the results are shown in Table 3 and graphed in Figure 4. My findings confirmed different effects of daf-2/daf-16 mutations in different tissues. The largest number of nematodes survived when DAF-16 was expressed in neuronal tissue as opposed to muscular (t = 13.87, p < 0.0001) and intestinal (t = 15.73, p < 0.0001) tissues. Additionally, DAF-16 expression in muscular tissues allowed more nematodes to survive compared to those with DAF-16 expressed in intestinal tissues (t = 5.14, p < 0.0001).


    Throughout my investigation I was attempting to determine which mutations in the daf-2 pathway, as well as which set of tissues would confer anoxia survival for C. elegans. Stress resistance is regulated through the DAF-16 transcription factor, which actually functions to initiate the stress response genes in the nematode. The daf-2 gene is responsible for making an insulin-like receptor, which is located on the outside of the cell. Under normal conditions, this receptor can sense a base-level of ligands (i.e. insulin molecules) which bind to the receptor and deactivate the DAF-16 transcription factor, whose default state is always active. Under stress conditions, the number of ligands decreases and the DAF-16 transcription factor is activated. This process is similar to keeping a computer plugged in an outlet; the constant source of power keeps the battery off, but when that power is taken away, the battery kicks in. The DAF-16 transcription factor localizes to the nucleus under stress conditions and transcribes genes that are responsible for stress resistance.

    A way to study the daf-2 gene is to induce mutations in it, which alters the insulin-like receptor, which, in turn, cannot transduce normal signals to DAF-16. This usually leads to DAF-16 activation, which then transcribes stress response genes. I wanted to determine which mutations and molecular processes were necessary for the anoxia survival that these mutants exhibited. By studying mutants and identifying which mutations lead to greater survival, we can then determine which genes are vital for stress survival, and employ this understanding toward developing therapeutic strategies.

    I studied five alleles of C. elegans daf-2 mutants, each of which had a mutation that disabled different parts of the insulin-like receptor. I chose these alleles based on how strong their stress resistance was from previous experiments (Gems et al., 1998). I chose daf-2(m41)daf-2(e1368), and daf-2(m577) from Class 1, and daf-2(e979) and daf-2(1391) from Class 2. All the mutants from both classes survived 3 days of anoxia, (compared with 1 day for the wild-type) showing that all the mutations I tested played a significant part in allowing the worm to survive anoxia (RQ1 & 2). I decided to increase the length of time in anoxia to 5 days, to see if some mutations were much better than others at allowing the nematode to survive anoxia. Only the daf-2(e1368) and daf-2(m41) mutants (Class 1) survived anoxia for 5 days (Figure 3). Both of these mutations are in the ligand-binding domain, which is the part of the receptor which binds to the ligands, and we see that if this part is damaged, in a specific place as specified by those mutations, the worm survives longer in anoxia. This indicates that these mutations are far more important than the other mutations in this pathway for regulating long-term stress resistance, especially anoxia survival.

    My results also showed that Class 1 mutants survive anoxia at greater percentages than Class 2 mutants (RQ3). These findings were consistent with earlier results which showed that Class 1 mutants had increased longevity compared to Class 2 mutants (Gems, et al., 1998). However, it was not known how anoxia was regulated through thedaf-2 pathway, and my novel finding was that the ligand-binding domain of Class 1 mutants had a disproportionate effect on anoxia survival, and increased survival up to 5 days.

    In addition to determining which genes might be important in anoxia survival, I was further interested in determining if certain tissues play more important roles in anoxia survival, as they did in increasing longevity (Libina, et al., 2004). I tested three mutants in which the DAF-16 transcription factor under the ges-1, unc-119, or myo-3 promoters was expressed in the intestinal, neuronal, or muscle tissue respectively. I observed that DAF-16 expressed solely in the neurons allowed C. elegans to survive in anoxia for longer periods of time compared to mutants with DAF-16 in muscles and intestines (RQ4). Furthermore, DAF-16 expression in muscular tissue provided higher survival percentages compared to intestinal DAF-16 expression (Figure 3). Additional research will need to be done to establish how signaling from the neurons influences survival. Determining if a combination of DAF-16 expression in several tissues increases the survival rate to even greater levels can give scientists a starting point for therapeutic strategies, and I am currently performing such a study.


    Through the use of a model organism, C. elegans, I was able to identify some of the genes that are responsible for long-term anoxia survival. I hypothesized that certain daf-2 alleles would survive anoxia at greater lenghts, and found that two alleles in particular,daf-2(e1368) and daf-2(m41) survived anoxia at high percentages after 5 days. Additionally, based on previous data on the insulin-like signaling pathway, I was able to deduce that the ligand-binding domain plays an important role in long-term anoxia resistance and is likely to be important in minimizing tissue damage during heart attacks and strokes. My experiments with anoxia survival of Class 1 and Class 2 mutants confirmed previous findings which showed that Class 1 mutants are better at resisting stress than Class 2 mutants. I discovered that some of the mechanisms governing anoxia survival and other stress responses are similar.

    In testing transgenic strains I found that DAF-16 expressed in the neurons only, allows C. elegans to survive anoxia at higher percentages compared with controls, and animals that had DAF-16 expression in other tissues. Past findings showed that DAF-16 in the intestine was enough to increase the longevity of the nematode, so in showing that DAF-16 in the neurons was more important in anoxia survival, I uncovered the subtle differences between the mecahnisms that regulate anoxia survival, and other stress responses. This finding is pertinent to future research to reveal how anoxia surivival is both similar and different from other stress responses, in order to combat human disesases. Additionally, this finding gives researchers of anoxic diseases in humans a place to start when elucidating therapies. Overall, my research was able to pinpoint genes in the insulin-like signaling pathway that are likely to increase human resistance to anoxic diseases through the FoxO pathway, and also show that the neurons are an important tissue when it comes to resisting anoxic stresses.


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    Table 1: Viabilities of Class 2 Alleles After 3 Days of Anoxia

    Strain Name Percent Survival Standard Deviation
    daf-2(e979) 87.91% 6.116
    daf-2(1391) 86.90% 6.770
    Wild-type (N2) 27.53% 7.039

    The survival rate of daf-2(e1391) and daf-2(e979) adult animals exposed to anoxia. Wild-type and daf-2 animals were exposed to 3 days of anoxia. This data is based on 6 independent trials, with 50 animals from each strain used for each trial. These results are based on N~300 animals.

    Table 2: Viabilities of Class 1 Alleles After 5 Days of Anoxia

    Strain Name Percent Survival Standard Deviation
    daf-2(e1368) 71.67% 4.956
    daf-2(m577) 0% 0
    daf-2(m41) 72.8% 15.99
    Wild-type (N2) 0 0

    The survival rate of daf-2(e1368), daf-2(m577), and daf-2(m41) adult animals exposed to anoxia. Wild-type and daf-2 animals were exposed to 5 days of anoxia. This data is based on 6 independent trials, with 50 animals from each strain used for each trail. These results are based on N~300 animals. 

    Table 3: Viabilities of Transgenic Mutants After 3 Days of Anoxia

    Strain Name N Percent Survival (SD) t-value p-Value against control
    312 3.673% (3.56) 2.5089 0.0124
    293 2.442% (2.10) 9.0767 <0.0001
    179 14.02% (12.32) 13.0353 <0.0001
    Wild-type (N2) 300 4.333% (2.9)  

    The survival rate of the 3 transgenic strains exposed to anoxia at the young adult stage. Wild-type and daf-2 animals were exposed to 3 days of anoxia. This data is based on 6 independent trials (4 trials for the punc-119::daf-16), with about 50 animals from each strain used for each trial. Statistical analysis was necessary to understand whether the differences in survival percentages were significant.

    Figure 1: Wild-type daf-2/daf-16 stress response activation

    Interactions between daf-2/daf-16 genes and stress response genes in wild-type C. elegans. This figure shows that without mutations in the daf-2 gene, stress response genes are inactive, and stay inactive until an environmental stress triggers the DAF-2 receptor to activate the DAF-16 transcription factor which then transcribes the stress response genes.

    Figure 2: Viability of Class 2 alleles after 3 days of anoxia

    The survival rate of daf-2(e1391) and daf-2(e979) adult animals exposed to anoxia. Wild-type and daf-2 animals were exposed to 3 days of anoxia. The data shown represents the tables shown previously, and represents at least 6 independent experiments with a total of 50 animals for each experiment. Error bars represent standard deviation.

    Figure 3: Viabilities of Class 1 alleles after 5 days of anoxia

    The survival rate of daf-2(e1368), daf-2(m577), and daf-2(m41) adult animals exposed to anoxia. Wild-type and daf-2 animals were exposed to 5 days of anoxia. The data shown represents at least 6 independent experiments with a total of 50 animals for each experiment. Error bars represent standard deviation.

    Figure 4: Viabilities of transgenic strains after 3 days of anoxia

    The survival rate of transgenic alleles in anoxia. These strains were tested in 3 days of anoxia and wild-type nematodes were used as controls. This graph represents at least 4 individual experiments with 50 animals for each experiment. Error bars represent standard deviation.