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First published online 11 January 2006
doi: 10.1242/dev.02232
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Department of Biology, McGill University, 1205 Dr Penfield Avenue, Montréal, Québec H3A 1B1, Canada.
* Author for correspondence (e-mail: richard.roy{at}mcgill.ca)
Accepted 1 December 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Insulin, TGF-ß, AMPK, PTEN, LKB1, Cell cycle, Lifespan, Dauer, Germ line, C. elegans
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The other two cells present in the primordium (Z2, Z3) are
the germ cell precursors and, unlike the divisions of the somatic gonadal
precursors, which are essentially invariant from one animal to another, Z2 and
Z3 do not demonstrate the same stereotypic division pattern and/or division
timing as they proliferate to form the germ lineage
(Kimble and Hirsh, 1979).
Even though the C. elegans germ line develops as a syncytium,
individual germline nuclei and their surrounding cytoplasm can be referred to
as germ cells (Hubbard and Greenstein,
2000). Sustained proliferation of the germ cell precursors and
their daughters during larval development requires signalling through a Notch
receptor. The Notch ligand (LAG-2) is expressed in the somatic DTCs, and
through the activation of a Notch receptor (GLP-1) present in the germ cells
it inhibits them from executing their alternative meiotic pathway, while
instructing them to proliferate (Kimble
and Crittenden, 2005). Initially, owing to the influence of a
nearby DTC, the germline stem cell population expands until the late L3 larval
stage. At this point, however, the proximal-most germ cells no longer receive
the Notch-dependent proliferative signal provided by the DTCs, and therefore
they execute the alternative meiotic pathway. In hermaphrodite animals, a
transient period of spermatogenesis is initiated, followed by a switch to
oogenesis in the L4 larval stage (Ellis
and Schedl, 2006).
Germline proliferation therefore proceeds uninterrupted in the distal germ
line during post-embryonic development, but can be pre-empted when
environmental conditions are sensed to be inappropriate to sustain
reproductive development during the L1 larval stage. Three parallel signals,
including reduced insulin-like signalling, can independently generate a
neuroendocrine signal that triggers a developmental switch, instructing the
larva to execute an alternative diapause-like stage referred to as `dauer'. In
anticipation of initiating the dauer pathway, the second larval (L2) stage is
extended (L2d), allowing the larva to prepare for nutrient deprivation by
slowing development and metabolism, while storing energy
(Riddle and Albert, 1997). One
important feature of this developmental switch is the progressive
establishment of a generalized cell cycle arrest that is maintained
thereafter, presumably to conserve resources, thus rendering the dauer larva
specialized for long-term survival.
The transcriptional targets of the downstream effectors that mediate dauer
development identified to date include genes involved primarily in metabolic,
antimicrobial and stress-response pathways
(McElwee et al., 2003;
Murphy et al., 2003;
Shostak et al., 2004).
Although much interest has been focused on the regulation of adult lifespan by
some of these factors, the key to the extreme longevity and developmental
quiescence of the dauer juvenile remains largely unclear. Most importantly,
how the three parallel signalling pathways (insulin-like, TGF-ß, cGMP)
that control dauer formation interact and presumably converge on the same
downstream targets in order to specify the behavioural, morphological,
physiological and metabolic changes associated with this stage is not well
understood.
Through our analysis of the mechanisms involved in controlling germ cell divisions during the establishment of developmental quiescence, we found that the independent signals that promote dauer formation converge to control the activity of the tumour suppressor gene daf-18/PTEN, an upstream component of the insulin-like signalling pathway. The regulation of germline proliferation, however, diverges from the canonical insulin-like targets Akt/PKB and daf-16/FOXO, but requires the tumour suppressor par-4/LKB1 and its downstream target AMP-activated kinase (aak-1, aak-2/AMPK) to appropriately arrest proliferation of the germline stem cell population in response to the environmental signals that induce dauer development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), unless
otherwise stated. The Bristol (N2) strain was used as wild type throughout.
The following alleles, rearrangements and transgenes were used. LGI:
daf-16(mgDf47, mgDf50), rrf-1(pk1417), szT1[lon-2(e678)].
LGIII: daf-2(e1370), daf-7(e1372), unc-32(e189), glp-1(oz112, q175,
e2141), unc-36(e251), pha-1(e2123). LGIV: daf-18(e1375, ok480).
LGV: qIs56[lag-2p::GFP; unc-119(+)],
akt-1(mg144), him-5(e1467), par-4(it47, it57). LGX: aak-2(rr48,
ok524), unc-1(e719), unc-7(e5), mec-4(u52), lin-15(n765), yDf1, szT1.
qEx308[lag-2p::LAG-2::GFP; rol-6(su1006)],
arEx645[pha-1(+); lag-2p::CFP::lacZ;
ceh-22::GFP] (Horvitz et al.,
1979).
aak-2(ok524) is a predicted molecular null allele in which part of
the catalytic domain is deleted; this introduces a premature stop codon
shortly after the breakpoint, thus eliminating the conserved regulatory domain
(Apfeld et al., 2004). The
deletion in daf-18(ok480) is predicted to remove part of the
C-terminal regulatory domain, while introducing a premature stop codon after
the breakpoint.
Genetic screen for regulators of germ cell proliferation during dauer development
daf-2(e1370) is a temperature-sensitive hypomorphic
allele of the C. elegans insulin-like receptor, which allows
reproductive development at the permissive temperature (15°C), but induces
dauer formation constitutively at the restrictive temperature (25°C)
(Albert and Riddle, 1988;
Kimura et al., 1997). The F1
progeny of EMS mutagenized daf-2(e1370); qIs56 L4 larvae were reared
at 15°C until the adult stage, at which point they were distributed five
per plate and up-shifted to 25°C. F2 dauer larvae were subsequently
screened for gonad enlargement using lag-2::GFP, the DTC expression
of which delineated the distal extremes of the dauer gonad. Using this
approach, we screened 3800 haploid genomes and isolated one mutant
(rr48) that demonstrated dauer germline hyperplasia and dauer
lethality. This mutant was outcrossed five times prior to any subsequent
analysis.
Mapping and cloning of rr48
Using conventional linkage analysis, followed by three-factor crosses,
rr48 was mapped to the far right of LGX. After crossing
daf-2; rr48 males into daf-2; unc-7 mec-4
hermaphrodites, we found 0/59 Mec non-Unc F2 recombinants that produced
rr48 F3 progeny, whereas 15/15 Unc non-Mec produced rr48 F3
animals, indicating that rr48 is tightly linked to, or to the right
of, mec-4, at the far right of LGX. The rr48 map position
was refined using transformation rescue. The YAC clone Y53B5, which is covered
by seven overlapping cosmids, rescued rr48. The cosmids were injected
in three overlapping pools of three, and the second and third pools both
rescued rr48. The cosmid T01C8 was common to both pools, and contains
five open reading frames. T01C8.1, a predicted C. elegans orthologue
of the mammalian 
2 catalytic subunit of AMP-activated kinase
(aak-2), was an obvious candidate as its yeast and mammalian
orthologues have been implicated in cell cycle/growth control
(Hardie, 2005). From T01C8, we
sub-cloned an 11.1 kb fragment (NcoI/PstI) containing
aak-2 into pGEM-T and injected it (10 ng/µl) into daf-2;
rr48 hermaphrodites, rescuing the dauer lethality phenotype. To
identify the molecular lesion in rr48, we cloned and sequenced
aak-2 cDNA from mutant animals. In three different clones obtained
from two independent RT-PCR reactions, we found a typical EMS-induced G/C to
A/T transition at position 1 of the predicted codon 208. This mutation is
predicted to substitute a conserved histidine with a tyrosine residue in a
highly conserved region of the catalytic domain. Furthermore,
aak-2(ok524) and aak-2(RNAi) phenocopied the dauer germline
hyperplasia and dauer lethality of rr48 mutants, confirming our
positional cloning results.
Staining
For whole worm DAPI (4',6-diamidino-2-phenylindole) staining, animals
were washed off plates and soaked in Carnoy's solution (60% ethanol, 30%
acetic acid, 10% chloroform) on a shaker overnight. Animals were then washed
twice in PBST (1xPBS + 0.1% Tween 20), and stained in 0.1 mg/ml DAPI
solution for 30 minutes. Finally, larvae were washed four times (20 minutes
each) in PBST, and mounted in Vectashield (Vector Laboratories) medium.
For extruded dauer gonad staining, gonads were dissected, fixed and stained
as described elsewhere (Arduengo et al.,
1998). Primary rabbit polyclonal anti-GLP-1, rabbit polyclonal
anti-P-granule (anti-PGL-1) and mouse monoclonal 1CB4, and secondary
anti-rabbit FITC and anti-mouse Texas-Red-conjugated (Invitrogen) antibodies
were used. DAPI was used as a counterstain.
Germ cell nuclei counts
DAPI staining was performed on a synchronized (by hatching in the absence
of food) population of L1 larvae plated and grown at 25°C until the
appropriate stage/time was reached, except for RNAi experiments, where adult
animals were allowed to lay eggs at 15°C overnight before the plate was
transferred to 25°C, and the adults killed. The total number of germ cell
nuclei per hermaphrodite gonad was then determined, based on their position
and nuclear morphology. Animals in which the proximal germ cells had developed
into spermatids were excluded from this analysis.
Dauer longevity assay
Because dauer larvae have a tendency to crawl off the bacterial lawn, and
often desiccate on the edges of the plate, we developed a novel method to
monitor the survival of individual dauer larva. Briefly, C. elegans
were synchronized and plated at 25°C. Three days later, 
10 dauer
larvae were randomly picked into a 20 µl drop of double-distilled
H2O suspended under a Petri dish cover. We refer to this system as
`the dauer trap'. A wet tissue was placed in the bottom of the dish to
maintain humidity, and the plate was sealed with Parafilm. Dauer longevity was
monitored daily, and survival was scored as moving response upon exposure to a
focused beam of 425-440 nm light. For the ablation experiments,
unc-1(e719) was introduced in the background of each strain to
prevent dauer larvae from crawling off the agar and survival was scored daily
as response to prodding.
Dauer recovery assay
Worms were synchronized and plated at 25°C for 50 hours. Dauer
larvae were then picked to fresh, pre-acclimated plates at 25°C in cohorts
of 100 individuals. Recovery was monitored daily. In
Fig. 2B, him-5(e1467)
was in the background of daf-7(e1372); aak-2(ok524).
All other techniques, such as microscopy
(Kostic et al., 2003), adult
longevity and dauer formation assays (both at 25°C)
(Hertweck et al., 2004), laser
ablation, aak-1 RNAi (Kostic et
al., 2003), and aak-2 RNAi
(Kamath and Ahringer, 2003),
were performed as previously described.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Hence, we reasoned that mitosis must be inhibited in the
germ line downstream of, or in parallel to, lag-2, by signals that
promote dauer formation.
To better understand how the dauer-associated cell cycle quiescence is
established in the germline stem cell population through compromised
insulin-like signalling, we performed a genetic screen to isolate mutants
defective in regulating germline proliferation during dauer development
triggered by reduced insulin-like signalling. We isolated one mutation
(rr48) that causes pronounced germline hyperplasia in insulin-like
receptor (daf-2) (Kimura et al.,
1997) mutant dauer larvae (Fig.
1D). Although pharyngeal and body radial constriction are
partially incomplete, daf-2; rr48 mutant dauer larvae
demonstrate all of the morphological and behavioural characteristics
associated with this stage, including SDS resistance
(Cassada and Russell, 1975)
(data not shown). By contrast, rr48 animals show no obvious
morphological, behavioural or reproductive phenotype during reproductive
development (data not shown).
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2-catalytic subunit of the
mammalian AMP-activated kinase (AMPK): aak-2 (AMP-activated kinase
2) in C. elegans. Biochemical studies have shown that AMPK
exists as a heterotrimeric complex composed of one catalytic -subunit,
and two regulatory subunits (Carling,
2004). The EMS-induced point mutation in aak-2(rr48) is
predicted to disrupt the catalytic activity of the 2-subunit, without
altering the regulatory domain. aak-2(rr48) heterozygous animals
display a dauer germline phenotype that is intermediate between the wild-type
and homozygous mutant situations, and this is not due to haplo-insufficiency
(Table 1, rows A-D).
Furthermore, aak-2(RNAi) partially suppresses the germline phenotype
of aak-2(rr48) mutants (Table
1, row E). Our analysis therefore suggests that
aak-2(rr48) behaves semi-dominantly in the germ line because of a
dominant-negative effect that most likely arises through interference with the
activity of the second C. elegans AMPK catalytic subunit:
aak-1 (AMP-activated kinase 1). Consistent with this, our
experiments indicate that aak-1 acts additively with aak-2
to inhibit germline proliferation during dauer development
(Table 2, rows F,J,L).
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In both daf-2 and daf-7 [TGF-ß mutant
(Ren et al., 1996)] dauer
larvae, germ cells arrest in interphase of the mitotic cell cycle
(Fig. 1F). The intensity of
DAPI-stained germ cell nuclei in daf-2 dauer animals is twofold
greater than that of the DTC (data not shown), suggesting a 4C DNA content,
consistent with a G2/M-phase arrest. Hence, during the dauer stage, germ cells
are not only mitotically quiescent, they are also blocked from initiating
their alternative meiotic program. During reproductive development,
inactivating mutations in the core components of the Notch signalling pathway
limit mitotic divisions in the germ line and cause premature meiotic entry,
resulting in the production of only a small number of sperm
(Austin and Kimble, 1987). To
test whether aak-2 also regulates the execution of the meiotic
program, we first examined whether germ cells could undergo meiosis if the
Notch-dependent meiotic block was removed from the germ line of dauer larvae.
The germ cells of daf-2 glp-1 larvae enter meiosis prematurely during
L2d, but arrest nonetheless in late stage meiotic prophase I in dauer
(Fig. 1G), indicating that
meiotic progression is blocked independently of Notch signals during this
stage. Remarkably, in daf-2 glp-1; aak-2 mutants, germ cells
progress through the entire meiotic program, including both meiotic divisions,
during dauer development and in fact, undergo spermatogenesis
(Fig. 1H-L). This suggests that
under these conditions, aak-2 ensures that gametogenesis is
appropriately coordinated with somatic development, and, in its absence, germ
cells can differentiate to sperm prematurely. We therefore conclude that
aak-2 contributes to mitotic and meiotic cell cycle regulation to
ensure quiescence of the germ cells under compromised insulin-like signalling
conditions during dauer formation.
aak-2 couples developmental arrest to lifespan extension
In addition to the observed germline hyperplasia, daf-2;
aak-2 dauer larvae die within 12-14 days
(Fig. 2A), unlike wild-type
dauer larvae, which are non-ageing and can survive beyond 70 days
(Klass and Hirsh, 1976). The
lethality of daf-2; aak-2 dauer larvae was not suppressed by
ablation of the germline precursors, or of the somatic gonad, or of both
(Fig. 3), suggesting that this
effect is independent of the germ line defect. Therefore, our data suggest
that the extreme lifespan extension characteristic of the C. elegans
dauer larva does not directly or solely result from the generalized cell
cycle/developmental arrest associated with this larval stage, but largely
requires aak-2 activity.
Conversely, compared with daf-7 mutants, which form dauer larvae
constitutively as a result of compromised TGF-ß function, daf-7;
aak-2 larvae exhibit a marked increase in recovery from dauer
(Fig. 2B). Because
aak-2 dauer larvae display both germ line and dauer longevity defects
when induced by either reduced cGMP, TGF-ß or insulin-like signalling, or
by crowding/starvation, or dauer pheromone
(Table 2, rows A-E,N-P;
Fig. 2A,B; data not shown),
aak-2 is likely to be required downstream of each of these pathways
to ensure appropriate dauer development. Furthermore, because insulin-like
signalling is necessary for dauer recovery
(Kimura et al., 1997;
Ogg et al., 1997), and because
reducing aak-2 function causes premature recovery in daf-7
dauer larvae, insulin-like signalling may negatively regulate aak-2,
which would act to promote dauer maintenance. In most organisms examined to
date, reduced insulin-like signalling causes adult lifespan extension
(Kenyon et al., 1993;
Kimura et al., 1997;
Lin et al., 1997;
Ogg et al., 1997). As the
genes involved in the regulation of dauer longevity may additionally affect
adult lifespan, we verified whether aak-2 mutations could also
antagonize the extension of adult lifespan in insulin-like mutants. Consistent
with this, aak-2 mutations suppress the lifespan extension of
daf-2 mutants considerably (Fig.
2C), indicating that aak-2 is required to potentiate the
effect of reduced insulin-like signalling on adult lifespan.
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; Kimura et al.,
1997; Ogg and Ruvkun,
1998; Paradis and Ruvkun,
1998). To directly test whether the level of insulin-like
signalling regulates germline proliferation through aak-2 in a
dose-dependent manner, we verified whether mutations that are known to
activate this signalling pathway downstream of the insulin-like receptor
(daf-2) could phenocopy the effect of aak-2 mutations on
germ cell proliferation at the onset of dauer. We therefore introduced a
hypomorphic mutation in the C. elegans PTEN orthologue
daf-18 (Ogg and Ruvkun,
1998), or a gain-of-function mutation in an Akt/PKB orthologue,
akt-1 (Paradis and Ruvkun,
1998), to partially upregulate insulin-like signalling in
daf-2 mutant larvae. daf-2; daf-18 and
daf-2; akt-1(gf) animals form dauer larvae that have 164.9%
and 43.4% more germ cell nuclei than do daf-2 animals, respectively
(Table 2, rows G,H), indicating
that efficient inhibition of germline proliferation by reduced insulin-like
receptor function occurs via the proper activation of daf-18,
inactivation of akt-1, as well as the activation of aak-2.
Interestingly, the effects of daf-18(lf) or akt-1(gf), and
aak-1/aak-2 inactivation on dauer formation and germline
proliferation are additive (Table
2, rows I,K,M), suggesting that these pathways work in parallel,
or that other factors, in addition to aak-1/aak-2, function
downstream of daf-18 and akt-1 to regulate dauer formation
and insulin-dependent cell cycle quiescence in the germ line.
Activity of the FOXO-like forkhead transcription factor daf-16, a
direct Akt/PKB target (Lee et al.,
2001), is believed to be responsible for all of the phenotypes of
reduced insulin-like receptor function
(Lin et al., 1997;
Ogg et al., 1997). We
nonetheless examined daf-16(0); daf-2 animals to determine
whether low insulin-like receptor activity would still result in reduced germ
cell proliferation even in the absence of daf-16. Although these
animals undergo reproductive development seemingly like wild-type animals, we
find that germline proliferation is slightly delayed in daf-16(0);
daf-2 young larvae when compared with wild-type or daf-16(0)
larvae (Fig. 4), suggesting
that daf-16-independent targets of the insulin-like receptor may also
affect germline proliferation. However, because the difference is minor
compared with that observed in the presence of a wild-type copy of
daf-16, such as in daf-2 mutants alone, our data confirms
that the regulation of germline proliferation by compromised insulin-like
signalling largely requires the activity of the transcription factor
daf-16. Therefore, it is likely that the regulation of aak-2
activity by the insulin-like signalling pathway largely occurs downstream of
daf-16, although it is impossible for the moment to rule out a
parallel mode of action.
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), however, several daf-16(0); daf-7 dauer
larvae did not maintain germ cell cycle quiescence and resumed reproductive
development (data not shown). Our data therefore suggest that reduced
TGF-ß signalling downregulates germline proliferation during dauer
development primarily through the activation of daf-18 and
aak-1/aak-2, whereas the inactivation of akt-1 and
the subsequent activation of daf-16 appear to be required to ensure
the proper maintenance, rather than the establishment, of germ cell cycle
quiescence in larvae with compromised TGF-ß signalling.
TGF-ß and insulin-dependent signals target aak-2 to regulate proliferation cell autonomously
Because the insulin-like and TGF-ß pathways demonstrate both cell
autonomous and cell non-autonomous functions
(Inoue and Thomas, 2000;
Wolkow et al., 2000), we
determined whether aak-2 activity is required for germ cell cycle
regulation in the tissues where these signalling pathways function to regulate
dauer formation, or within the germ line proper. To address this question, we
studied the response of rrf-1 mutants to aak-2(RNAi). rrf-1
mutants are resistant to dsRNA-mediated interference in the soma, whereas they
remain RNAi-sensitive in the germ line
(Sijen et al., 2001).
aak-2(RNAi) performed in both rrf-1; daf-2 and
rrf-1; daf-7 animals recapitulated the germline hyperplasia
typical of daf-2; aak-2(RNAi) and daf-7;
aak-2(RNAi) dauer larvae (Table
3, rows A-H), indicating that aak-2 is required cell
autonomously (within the germ cells) to regulate germline proliferation in
response to compromised insulin-like and TGF-ß signalling, during dauer
development.
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; Woods et al.,
2003). In humans, inactivating mutations in LKB1 are responsible
for most cases of Peutz-Jeghers Syndrome, a rare dominantly inherited disorder
characterised primarily by predisposition to benign and malignant tumours in
many organ systems (Hemminki et al.,
1998; Jenne et al.,
1998). We reasoned that if LKB1 was required for optimal AMPK
function, inactivating mutations in the C. elegans LKB1 orthologue
par-4 (Watts et al.,
2000) should phenocopy the effect of reduced
aak-1/aak-2 activity. par-4 mutations indeed
phenocopy the dauer germline hyperplasia caused by the inactivation of both
aak catalytic isoforms (Table
2, rows J,L; Table
4, rows D-G), indicating that par-4 is required to
suppress proliferation of the germline stem cell population in response to
reduced insulin-like or TGF-ß signalling levels. If the germline
hyperplasia in par-4 mutant dauer larvae results from defective
aak activation, the loss of aak-1 and/or aak-2
activity should not enhance the effect of a strong par-4(it57)
inactivating mutation (Watts et al.,
2000). However, only aak-1(RNAi) was not significantly
additive to par-4(it57) (Table
4, rows I,L). In fact, while daf-2; par-4 dauer
larvae are long-lived when compared with daf-2; aak-2
animals (Table 4, rows A-E),
par-4 enhances the dauer germline and lifespan phenotypes of
aak-2 mutants (Table
4, rows H,J-K), suggesting that par-4 and aak-2
function, at least in part, in an additive manner, and that aak-2
activity may not absolutely rely on par-4 to control germ cell cycle
and lifespan during compromised insulin-like signalling conditions. Moreover,
our data suggest that par-4 may target other factors along with
aak-1/aak-2 to control germline proliferation under these
dauer-promoting conditions.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
plant, yeast and mammalian systems, the AMPK orthologues act as `metabolic
master switches' to appropriately reset gene expression for survival on
alternative energy sources (Carling,
2004; Hardie et al.,
1998). The lifespan extension that is associated with
developmental arrest and/or compromised insulin signalling in most organisms
is therefore likely to require a substantial switch to alternative metabolic
pathways, which is triggered predominantly through the activation of AMPK. We have demonstrated that daf-18/PTEN, par-4/LKB1 and aak-1, aak-2/AMPK are crucial factors commonly required for the regulation of germline proliferation in response to the neuroendocrine signal generated as a result of compromised insulin-like and/or TGF-ß signalling (Fig. 5), whereas daf-16/FOXO appears, at least in part, dispensable. Our data is therefore consistent with a model in which par-4 and aak-1/aak-2 function together with daf-18, and partially in parallel with daf-16. Namely, the efficient suppression of germ cell cycle progression during early larval development requires either compromised TGF-ß signalling or daf-16 activity. Although we provide evidence that aak-2 is required in the germ line to control proliferation in response to these signalling pathways, it would be of great interest to determine the cellular requirement of each of the players that link the insulin-like and/or TGF-ß-dependent neuroendocrine signals to the regulation of aak-2 activity within a target tissue.
Together, these results complement/modify our current model of cell
cycle/growth control, in which growth factors are believed to function through
Akt/PKB (which is negatively regulated by PTEN), while intracellular energy
stress is thought to work in parallel, activating AMPK through LKB1, then
converging on the TSC/mTOR pathway to cell autonomously regulate growth and
division (Corradetti et al.,
2004; Shaw et al.,
2004). Our work indicates that the insulin and TGF-ß
signalling pathways independently converge on both tumour suppressors, PTEN
and LKB1, which then mediate cell cycle control under stress conditions.
|
) and AMPK (Corradetti et
al., 2004; Shaw et al.,
2004); however, evidence that mutations in AMPK recapitulate the
proliferative defects associated with LKB1 mutations has up to now been
lacking. Our findings provide strong genetic evidence that the aberrant cell
cycle regulation associated with Juvenile Polyposis Syndrome, Cowden's Disease
and Peutz-Jeghers Syndrome, which are caused by mutations in genes involved in
TGF-ß signal transduction, in PTEN and in LKB1, respectively
(Waite and Eng, 2003;
Wirtzfeld et al., 2001), may
result from defects at different levels of a single genetic pathway that
unifies general growth factor levels to the regulation of cell proliferation
via AMPK regulation. |
ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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25 for each
point. (F) In both daf-2(e1370) and daf-7(e1372)
larvae, germ cells arrest in mitotic interphase during dauer (arrowhead), as
shown in a DAPI-stained, extruded daf-7(e1372) dauer gonad.
(G) In daf-2(e1370) glp-1(e2141) dauer larvae, germ cell
nuclei arrest in late stage meiotic prophase I, the nuclear size and
chromosome morphology being consistent with the pachytene stage (inset).
(H) Germ cells often (26/30 animals) progress further through meiosis
in daf-2(e1370) glp-1(e2141); aak-2(rr48) dauer larvae, and
their chromosomal morphology closely resembles that of spermatids (arrow).
Similar defects were observed in daf-2(e1370) glp-1(e2141);
aak-2(ok524) (19/40 animals) and in daf-2(e1370)
glp-1(e2141); par-4(it57) (25/30 animals) dauer larvae.
(I-L) Extruded daf-2(e1370) glp-1(e2141); aak-2(rr48)
dauer gonad. Asterisks indicate germ cells undergoing spermatogenesis. Cells
were stained with (I) DAPI (blue); (J) anti-P-granule (green), a germ cell
marker that is lost during spermatogenesis
(Gruidl et al., 1996
