impinge on gene expression of the affected individuals [5–8]. More-
over, these changes were further correlated with an increased fre-
quency of various disorders that were not observed in siblings that
were conceived during periods when adequate nutrition was acces-
sible [5, 9, 10].
Although the small free-living nematodeC. elegansdoes not
possess the enzymes required for cytosine methylation, changes in
histone modification have been linked to heritable transgenera-
tional phenotypes [2, 11–14]. Recently, our group described a
requirement for the AMP-activated protein kinase (AMPK) in
blocking specific histone modifications during periods of acute
starvation [15]. In its absence, a critical histone modification is
aberrantly written onto the chromatin, thereby earmarking these
regions of the genome as genes that should be, albeit inappropri-
ately, actively expressed. This dysregulation of gene expression
ultimately disrupts the integrity of the germ cells, which transmit
this information to successive generations, often becoming pro-
gressively worse with each subsequent generation.
To assess this multigenerational effect of reducing AMPK func-
tion in the early L1 larval stage, we designed a means of tracking
animals throughout several generations to assess any reproductive
defects that arise following the initial period of starvation. We
describe here how we use three different methods of tracking to
test different patterns of inheritance following the starvation during
the L1 diapause. The following sections describe an adaptable
means of assessing transgenerational phenotypes in C. elegans
with a particular focus on the defects that arise in the germ line
downstream of acute starvation using animals wherein both cata-
lytic subunits have been disrupted and therefore lack all AMPK
signaling. The same multigenerational analyses could easily be
modified to determine various other parameters such as metabolic
signatures and/or other physiological states of the AMPK mutant
descendants by ensuring that the starting populations will provide
enough animals at each generation to perform the desired assays in
a statistically significant manner.
AMPK null mutants (aak-1;aak-2) are viable if they are not
subjected to starvation or other stresses, and they are fertile if they
are not stressed during development. Therefore, hermaphrodites
can be maintained on minimal bacterial agar plates (NGM medium)
seeded with a thin layer ofE. coli(strain OP50) until they become
gravid (full of embryos) adults. Gravid adult hermaphrodites can
then be subjected to alkaline/hypochlorite treatment to dissolve
the animals, leaving the embryos intact. The embryos are washed
three times in M9 buffer and then placed in a minimum volume of
M9 buffer for incubation overnight to hatch the animals (details
below). These emergent first larval stage (L1) animals will survive in
the absence of food for approximately 2 weeks in a developmentally
quiescent state that is referred to as the L1 diapause. In this state,566 Emilie Demoinet and Richard Roy