AMPK Methods and Protocols

3.2
Transgenerational
Defects

3.2.1 Multigenerational
Analysis of Brood Size

Both sterility and brood size can be assessed at every generation

following the initial L1 diapause. It requires that the investigator

maintains an orderly array of plates that represent the progeny of

individual parents that were subjected to starvation at the L1 stage.

In our analysis of AMPK in the regulation of germ cell integrity, we

noted that fertile P 0 animals fell into two classes: those that gener-

ated a normal brood size and those that appeared compromised

(smaller brood size than wild type). Curiously, even if the parents

appeared normal, the next generation often had a reduced brood

size even when they were never subjected to starvation, as the initial

event is documented through epigenetic changes mediated

through histone modifications. We therefore monitored pheno-

typic defects in the AMPK mutants that were subjected to the L1

diapause in successive generations according to three different

methods of multigenerational analysis (Fig.2).

l Method 1: assuming that the animals that were most affected by

the acute starvation will always segregate the most affected

Method 1

single animals from parents that are always affected

Method 2

single animals from parents that are initially unaffected(at P 0 )

then always affected

... [P 0 ]

Affected Unaffected

Brood size <100, Ste Brood size ≥ 100

[P 0 ]

Randomly choose 4 platesAffected

Isolation of 30-100 L4 larvae per plate

... Fn=120-400^1

Development and phenotyping

... [F 1 ]

Aff Aff Unaff Aff Aff. Unaff.

Randomly choose 4 platesAffected

Isolation of 30-100 L4 larvae per plate,

development and phenotyping

[F 2 ]

n=120-400

... [Fn=120-400x]

Randomly choose 4 platesUnaffected

Isolation of 50 L4 larvae per plate

...

Development and phenotyping

...

Aff Aff Unaff. Aff Aff. Unaff.

Randomly choose 4 platesAffected

Isolation of 50 L4 larvae per plate,

development and phenotyping

...

[P 0 ]

F 1

n=200

[F 1 ]

[F 2 ]

n=200

[Fx]

n=200

F 1 L1

immunostaining

F 2 L1

immunostaining

Fx-1L1

immunostaining

Fig. 2Transgenerational analysis of animals subjected to acute starvation in the L1 diapause stage. We use
three distinct methods to test the degree of transgenerational impact following 3 days of starvation. Methods
1 and 2 differ in the reproductive phenotypes of the parents to be analyzed. Method 3 is only different from
Method 2 through continuous selection of L4 progeny that arises from unaffected parents at each generation.
Any generation of interest can be tested for histone modifications or other biological parameters by performing
alkaline hypochlorite treatment (bleaching) followed by immunostaining of the emergent L1 larvae. Reproduc-
tive fitness based on brood size of the parents is used to classify them as “affected (Aff)” or “unaffected” by
the initial starvation. Fxrefers to filial generation; Ste is sterile

572 Emilie Demoinet and Richard Roy