Discovery Talk Questions and Answers: A Genetic Control Circuit for Aging

Cynthia Kenyon
Assessments created by Dr. Delquin Gong

Questions

In the paper, Dr. Kenyon and colleagues designed their experiments using temperature-sensitive mutant worms.
1. What does it mean to have a temperature-sensitive mutation?
2. How do you induce the mutation?
In the paper, Dr. Kenyon compares their daf-2 mutants with wild-type worms as well as dauer worms.
1. What is a dauer?
2. Why are dauer worms useful controls?
Strikingly, the daf-2 mutants experienced a much longer lifespan than their wild-type counterparts. What else was striking about daf-2 mutants?
Which of the following behaviors/characteristics were compared between the daf-2 and the wild-type animals (select all that apply)?
1. Movement when stimulated with an eyelash
2. Physical size of the animals
3. Rate of feeding
4. Reproductive capability
5. None of the above
In the paper, Dr. Kenyon and colleagues compared the ‘pumping’ behavior of the daf-2 mutants to the wild-type worms (Fig 2a, b). They find that aging animals had more sporadic episodes of pumping.
1. How do they interpret this observation?
2. Suggest an experiment to test this hypothesis using the models and supplies used in other experiments in the paper?
In her talk, Dr. Kenyon mentions that there was already one gene that was known to affect the lifespan of C. elegans, and when this gene was mutated, the worms could live 50% longer but were also not very fertile. Evolutionary biologists posited that “it was possible that the reason they live long was that they didn’t have to channel all of their resources into reproduction, and so they would have more to live longer (2:45).” Dr. Kenyon didn’t necessarily think so.
1. In the paper, how did Dr. Kenyon and her colleagues directly test this hypothesis?
2. What is your interpretation of the data for the daf-2 mutants obtained?
In the list below, select other daf mutants, but that did not show any longer lifespan than wild-type worms. [Paper]
1. Daf-3
2. Daf-7
3. Daf-11
4. Daf-14
5. None of the above
In her talk, Dr. Kenyon mentioned that there is another daf mutant, daf-16, that is required for longer lifespan. Based on the data in the paper, which of the following describe the relation between daf-2 and daf-16? (Select all that apply).
1. Daf-2 and daf-16 belong in a linear pathway promoting dauer formation.
2. Daf-2 lies downstream of daf-16 in a pathway promoting extended lifespan.
3. Daf-2 mutations reduce daf-16 activity to promote extended lifespan.
4. None of the above
According to Figure 4, the daf-16(m26) single mutant displays a normal lifespan indistinguishable from wild-type animals. Suggest an experiment that would reveal the role of daf-16 in extending lifespan. [Paper]
In her paper, Dr. Kenyon surmises on the cellular function of daf-2, and then mentions in her talk that Gary Ruvkun’s lab cloned the daf-2 gene.
1. What does this gene encode, and what might be its cellular function based on its similarity to other known receptors?
2. Given daf-2’s potential cellular function, what is one way of extrinsically mimicking a loss of daf-2 activity?
Dr. Kenyon mentions that the human gene corresponding to daf-16 has been identified. What is the name of this family of genes?

Answers

1. a. A temperature-sensitive mutation is a conditional mutation “that has wild-type (or less severe) phenotype under certain “permissive” environmental conditions and a mutant phenotype under certain “restrictive” conditions” (Source: http://en.wikipedia.org/wiki/Mutation)
1. b. Temperature-sensitive mutant worms express a mutated gene, whose product is stable at low temperatures. Worms are grown at this “permissive” temperature until the appropriate developmental stage and then shifted to the higher, non-permissive temperature. The proteins then become unstable and non-functional, inducing a loss-of-function phenotype.
2. a. Dauer describes an alternative developmental stage of nematode worms, particularly Caenorhabditis elegans whereby the larva goes into a type of stasis and can survive harsh conditions. (Source: http://en.wikipedia.org/wiki/Dauer_larva)
p. 461: “Dauers are developmentally arrested animals that are small, thin and sexually immature larval form.”

2. b. Because they are also long-lived animals, they are useful positive controls in studies when searching for long-lived mutants.
Daf-2 mutants did not age and then stay old, their rate of aging appeared too slow.Video (4:40): “So, amazingly enough he set out to look for long lived mutants and he found one. He found that mutants that reduce the activity of a gene called daf-2 double the worm’s lifespan. Here you see in black the lifespan of a normal worm, so by the end of 30 days, or about a month or so, all the worms are dead. But as you see in red, the daf-2 mutant lives twice as long as normal, even more than twice as long. The thing that was the most cool about this, was that the worms didn’t just go into the nursing home and hang on, they were actually aging more slowly than normal.”
p. 463: “Daf-2 mutants did appear different from wild-type adults in at least two respects. First, they grew to adulthood more slowly than wild-type, talking 3 days rather than 2 at 20°C.”
a-c. All of these behaviors/characteristics were assessed as follows:
a. Movement when stimulated with an eyelash:

Fig. 2c (p. 463): “We investigated the what fraction of animals were moving actively on their plates, or began to move across the plates when touched with an eyelash.”

b. Rate of feeding:Fig. 2a and 2b (p. 463): “C. elegans ingests food by periodic contraction of its pharynx, called ‘pumping’. To determine the per cent of live animals pumping (a), each animal was examined twice within a 1h period for at least 30s each time. Average pumping rate (b) was determined by measuring pumping rates of at least five randomly selected animals that were pumping (…)”.

c. Reproductive capability:

Fig. 3: “The lifespan of the C. elegans hermaphrodite is not affected by the production of germ cells or progeny”
a. Dr. Kenyon and colleagues posit that the decreased pumping rates may reflect a decreased requirement for food when production of progeny decreases
p. 463: Fig 2 legend: “As both wild-type and daf-2 mutants aged, their pumping became sporadic. Often the animals lay still when first observed, but when stimulated, began to move across the plates, and often to pump. Individuals not pumping on one day would sometimes be pumping a few days later. Thus it seems likely that whereas young adults pump continuously, older animals (including mutant and wild-type) pump intermittently until close to death. It is possible that the decrease in pumping rate which occurs with similar kinetics in wild-type and daf-2 animals, is not due to senescence, but instead reflects a decreased requirement for food when production of progeny ceases.”

5. b. One way we can test this hypothesis is to measure the pumping rates of the daf-2 mutants and wild-type worms in which germ- and gonad-cell precursors were laser-ablated as described later in the paper (Fig. 3, p.463). This would allow the investigators to compare the pumping rates for animals that never had to expend energy for progeny production.
a.
1. They showed that daf-2 mutants had only slightly smaller brood sizes than wild-type.

p. 461: “Furthermore, at 20°C, where its lifespan was twice that of wild type, daf-2(e1370) animals had nearly normal brood sizes (212±36 for daf-2(e1370) (n=29); as compared to 278±35 for wild-type animals grown in parallel (n=19)).

2. To do this, They used laser beams to selectively destroy the cells that would become germ cells and gonads, so that worms could not reproduce when they matured, and looked at the lifespan of these worms.

p. 463, Fig. 3:
“The germ-cell precursors, Z2 and Z3, as well as the precursors of the somatic gonad, Z1 and Z4, were ablated in 27 newly hatched N2 hermaphrodites using standard procedures. Mean lifespans were 18 days (s.d.=6) for ablated animals (n=27) and 17 days (s.d. = 5) for N2 controls (n=27). These survival curves were not significantly different from one another.”

6. b. The authors do not show the data for the laser-ablated daf-2 mutants. Instead, they state that daf-2 mutants continued to exhibit the longevity phenotype despite the inability to produce progeny (Fig 3).
p. 463, Fig. 3:
“The lifespan of the C. elegans hermaphrodite is not affected by the production of germ cells or progeny. (…) The act of ablation itself was unlikely to affect lifespan, because daf-2 mutants still expressed the full longevity phenotype following ablation (data not shown).”
b, c, dp. 461: “The lifespans of daf-11, daf-7 and daf-14 adults were not noticeably different from wild type (Fig. 1 legend).”
p. 462: “The lifespans of the remaining animals were not significantly different from the wild type (data not shown).”
ab is incorrect because daf-16 is downstream of daf-2,
c is incorrect because daf-2 mutations increase daf-16 activity.
Possible Answers:1. Rather than bypass the decision to enter the dauer stage at L1, shift the wild-type and daf-16 mutant worms to non-permissive temperature at or before the L1 stage and determine whether daf-16 mutants extend their lifespan.
2. Shift all the animals at later stages to bypass the decision to enter dauer stage, and subject the animals to food restriction to determine whether the daf-16 mutants extend lifespan beyond that of wild-type animals.
a. The daf-2 gene encodes a hormone receptor. It has similarity to the human receptor for insulin as well as the insulin-like growth factor (IGF) receptor family, whose function is to regulate nutrient uptake and control cell growth, respectively. It is likely that daf-2 is also involved in regulation of nutrient uptake and cell growth.Video (7:39): “So what is this gene, what is this daf-2 gene? Well the Gary Ruvkun lab cloned the gene, and they found that daf-2 encodes a hormone receptor. So that means hormones control aging, and not only that, it is similar to receptors that we knew about, number one, the human receptor for insulin, which is a hormone that controls nutrient uptake, and also it was similar to a hormone called IGF-1 which controls growth, and these hormones are similar to one another that is, it was similar to the receptor for these hormones.Other Reference: Cynthia Kenyon, The genetics of aging. Nature 464(7288), 504-12 (2010) p. 504: “In C. elegans, mutations that decrease the activity of daf-2, which encodes a hormone receptor similar to the insulin and IGF-1 receptors, more than double the lifespan of the animal, and mutations affecting the downstream phosphatidylinositol 3-kinase (PI(3)K)/ AKT/PDK kinase cascade extend lifespan as well.” http://kenyonlab.ucsf.edu/Kenyon_2010_Nature_rev.pdf

10. b. Limiting food/food restriction.

p. 462 and p.464: “Lifespans of mammals, and, to a limited extent, C. elegans, can be increased by food limitation. It is possible that daf-2 mutations elicit an internal signal also generated by food limitation, which can extend lifespan without substantially affecting fertility or general activity.”
The mammalian orthologs of the daf-16 gene are the FOXO (Forkhead box O) family of transcription factors.
Video (9:08): “We also found a gene called daf-16 is needed for the long lifespan, and humans have this gene, and it’s called FOX-O and it turns out that FOX-O variants are associated with exceptional longevity in populations all around the world.”

Other Reference: Cynthia Kenyon, The genetics of aging. Nature 464(7288), 504-12 (2010)