C. elegans AMPKs promote survival and arrest germline development during nutrient stress

Abstract

Mechanisms controlling development, growth, and metabolism are coordinated in response to changes in environmental conditions, enhancing the likelihood of survival to reproductive maturity. Much remains to be learned about the molecular basis underlying environmental influences on these processes. C. elegans larvae enter a developmentally dormant state called L1 diapause when hatched into nutrient-poor conditions. The nematode pten homologue daf-18 is essential for maintenance of survival and germline stem cell quiescence during this period (Fukuyama et al., 2006; Sigmond et al., 2008), but the details of the signaling network(s) in which it functions remain to be elucidated. Here, we report that animals lacking both aak-1 and aak-2, which encode the two catalytic α subunits of AMP-activated protein kinase (AMPK), show reduced viability and failure to maintain mitotic quiescence in germline stem cells during L1 diapause. Furthermore, failure to arrest germline proliferation has a long term consequence; aak double mutants that have experienced L1 diapause develop into sterile adults when returned to food, whereas their continuously fed siblings are fertile. Both aak and daf-18 appear to maintain germline quiescence by inhibiting activity of the common downstream target, TORC1 (TOR Complex 1). In contrast, rescue of the lethality phenotype indicates that aak-2 acts not only in the intestine, as does daf-18, but also in neurons, likely promoting survival by preventing energy deprivation during L1 diapause. These results not only provide evidence that AMPK contributes to survival during L1 diapause in a manner distinct from that by which it controls dauer diapause, but they also suggest that AMPK suppresses TORC1 activity to maintain stem cell quiescence.

Keywords: AMPK; Diapause; Stem cell.

Fig. 1.. The aak genes promote survival during L1 diapause.

(A) Simplified signaling network integrating the IIS, AMPK, and TORC1 pathways (reviewed by Zoncu et al., 2011). Gene names of C. elegans homologues are indicated in parentheses. (B) Survival curves of indicated mutants during L1 diapause in M9 medium. (C) Viability of ampk mutants in M9 medium with or without the indicated energy source after 9-day L1 diapause. In B and C, the average of at least three independent experiments is reported and error bars indicate s.e.m. >100 animals were scored for each time point per experiment.

Fig. 2.. The aak genes act redundantly to maintain germline stem cell quiescence during L1 diapause.

(A) Time course of germline stem cell proliferation during L1 diapause. Animals were treated as described in Materials and Methods. ≥50 animals were scored for each time point per experiment. The average number of germ cells was determined for each time point in each of three experiments, and the average of these was plotted in the graph with error bars indicating s.e.m. (B) Germline precursors divide in ampk mutants during L1 diapause. Fluorescent micrographs of wild-type and ampk mutant germlines fixed and stained with DAPI (blue) and anti-PGL-1 (green) polyclonal antibody (Kawasaki et al., 1998) after 7-day L1 diapause. Individual germ cells are indicated by arrowheads. Scale bar, 5 µm. (C) L1 diapause causes sterility in ampk and daf-18 mutants. Animals were returned to food following a 5-day diapause and animals were singled when they were late L4s/young adults and their survival and fertility was scored 5 days later. Most ampk and about half of daf-18 mutant adults were sterile. Animals scored as “dead” died without producing progeny, usually by rupturing at the vulva. (D) Fertility was not impaired when ampk and daf-18 mutants were grown continuously in nutrient-rich conditions. >35 animals were scored for each time point per experiment. Animals in L1 diapause were cultured in M9 medium plus ethanol. In C and D, the average of three independent experiments is plotted with error bars indicating s.e.m.

Fig. 3.. aak-2 acts in distinct tissues to regulate survival and germline quiescence during L1 diapause.

(A) Effects of tissue-specific aak-2 expression on the survival of ampk mutants during L1 diapause. Here and in (B) and (C), the average of at least three independent experiments ± s.e.m. is plotted. The Y-axis indicates the transgenes tested for rescuing activity, and the tissue in which each promoter drives expression is indicated in parentheses. The animals carrying the transgenes were identified by GFP expression. (B) Effects of tissue-specific aak-2 expression on germline proliferation of ampk mutants during L1 diapause. The average number of germ cells after 7-day L1 diapause is plotted. In (A) and (B), ≥35 animals were scored for each experiment. (C) Effects of tissue-specific daf-18 expression on the survival of daf-18 mutants after 5-day L1 diapause. ≥100 animals were scored for each experiment. *P<0.01 for Student's t-test. “Mock” indicates analysis of animals transgenic for co-injection markers only.

Fig. 4.. TORC1 activity mediates ectopic germ cell proliferation in ampk and daf-18 mutants.

(A,B) Effects of TORC1 inhibition on the average number of germ cells in ampk (A) and daf-18 (B) mutants after 5-day L1 diapause. (C,D) Genetic elimination of Rag activity in ampk and daf-18 mutants. Genotypes are listed on the Y-axis and alleles used were as follows: aak-1(tm1944), aak-2(ok524), raga-1(ok386), ragc-1(tm1974), and daf-18(ok480). Number of animals scored in each experiment: (A) ≥24, (B) ≥10, and (C,D) ≥35. The average number of germ cells per genotype was determined for each experimental trial, and the average of three trials is plotted in the graph with error bars indicating s.e.m. *P<0.01 for Student's t-test.