metabolism is maintained at a low level, as is overall gene expres-
sion. Cell divisions do not occur, nor do L1 specific cellular move-
ments/migrations take place while the animal has not met an
appropriate food/energy contingency to trigger the onset of post-
embryonic development. The initiation of development can occur
at almost any point during this period by placing animals in the
presence of a food source, which in the laboratory consists of a lawn
ofE. colibacteria (here OP50, but other strains are also effective).
Ingesting the food is sufficient to trigger escape from the diapause
and the initiation of the larval developmental program. Animals
that are maintained for longer durations in the L1 diapause can
initiate development after feeding, but their progression is often
irregular, and they often give rise to sterile adults. Unlike AMPK
mutant animals, these starved wild-type animals do not transmit
these defects in a heritable manner, suggesting that AMPK plays a
critical role during the L1 diapause to adjust to the nutrient/energy
challenge associated with starvation and to maintain the integrity of
the primordial germ cells that will give rise to all the germ cells and
consequently the gametes, in the adult.
Here we describe how to quantify the various defects that arise
due to starvation in these AMPK mutant animals. We detail our
methods of examining multigenerational phenotypes that may arise
in animals that were subjected to acute starvation. In addition, we
provide a well-established protocol to analyze and quantify chro-
matin modifications that occur in the germ line using commercially
available antibodies. Although we focus on the germ line in
C. elegans, our methods can be easily adapted for detailed analysis
of somatic changes in gene expression or metabolism that may
occur in a similar transgenerational manner.2 Materials
The following solutions should be prepared in advance:- Alkaline hypochlorite (bleaching solution, 50 mL): 1.2% (v/v)
sodium hypochlorite (household bleach), 0.5 M KOH. This
should be prepared just prior to use in an amber 50 mL Falcon
tube to minimize exposure to light and hence loss of efficiency. - M9 buffer: 22 mM KH 2 PO 4 ,42mMNa 2 HPO 4 ,86mM
NaCl, 1 mM MgSO 4. - Ice-cold methanol (100% methanol kept at 20 C).
- Coating solution for poly-L-lysine coated slides: 0.1% (w/v)
poly-L-lysine, 0.1% (w/w) gelatin, 0.01% (w/w) chromoalum,
Milli-Q H 2 O (prepare 1 mL). - 2fixative solution: open a fresh 10 mL vial of 16% formalde-
hyde, and dilute to a final concentration of 4% formaldehyde
Quantifying Starvation-inducible Transgenerational Defects 567