DAF-2 and ERK Couple Nutrient Availability
to Meiotic Progression
during Caenorhabditis elegans Oogenesis
Andrew L. Lopez III,1,2Jessica Chen,1,2Hyoe-Jin Joo,1Melanie Drake,1Miri Shidate,1Cedric Kseib,1and Swathi Arur1,*
1Department of Genetics, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA
2These authors contributed equally to this work
Coupling the production of mature gametes and
fertilized zygotes to favorable nutritional conditions
improves reproductive success. In invertebrates,
the proliferation of female germline stem cells is
regulated by nutritional status. However, in mam-
mals, the number of female germline stem cells is
set early in development, with oocytes progressing
through meiosis later in life. Mechanisms that couple
later steps of oogenesis to environmental conditions
remain largely undefined. We show that, in the pres-
ence of food, the DAF-2 insulin-like receptor signals
through the RAS-ERK pathway to drive meiotic pro-
phase I progression and oogenesis; in the absence
of food, the resultant inactivation of insulin-like
signaling leads to downregulation of the RAS-ERK
pathway, and oogenesis is stalled. Thus, the insu-
lin-like signaling pathway couples nutrient sensing
to meiotic I progression and oocyte production in
C. elegans, ensuring that oocytes are only produced
under conditions favorable for the survival of the re-
To survive and propagate, organisms must respond to changes
in environmental conditions by altering their physiology and
behavior (Hietakangas and Cohen, 2009; Neufeld, 2003). Repro-
ductive development is particularly well tuned to changes in
environmental conditions. Reproductive needs are often coordi-
nated with energy requirements and dictated by environmental
conditions. For example, in C. elegans, larvae obtain sexual
maturity rapidly in normal environmental conditions due to acti-
vation of insulin signaling, but, in harsh conditions, the larvae
arrest in a sexually immature stage and enter an alternate state
of development: the dauer pathway (Antebi et al., 2000; Kenyon
et al., 1993). Changes in nutrient availability also impact verte-
brate reproductive development and success. For example,
work with cows, pigs, and sheep indicate that poor nutritional
status reduce oocyte quality and fecundity (Fouladi-Nashta
et al., 2007; Papadopoulos et al., 2001). Also, in humans, insulin
triggers the insulin growth factor receptor (IGFR1) to induce
progesterone secretion, which, in turn, promotes the maturation
of ovarian follicle cells and normal female fertility (Poretsky et al.,
1999; Silva et al., 2009).
In Drosophila and C. elegans, mechanistic studies indicate
that insulin signaling links nutritional conditions to the prolifera-
tion rate of germline stem cells (Drummond-Barbosa and Spra-
dling, 2001; Hsu and Drummond-Barbosa, 2009; Michaelson
et al., 2010). For example, in flies, a protein-rich diet appears
to induce the secretion of insulin-like peptides from the brain,
which act systemically to activate insulin signaling in remote
tissues (Colombani et al., 2003). In the ovaries, activation of
the insulin signaling pathway increases the division rate of both
somatic and germline stem cells, promotes cell survival, and in-
creases vitellogenesis, a process by which oocytes uptake yolk
during ovarian follicle maturation (Drummond-Barbosa and
Spradling, 2001; LaFever and Drummond-Barbosa, 2005). In
worms, in response to nutrient-replete conditions, activation of
the DAF-2 insulin-like receptor also enhances germline stem
cell proliferation in the female germline during larval stages and
in certain tumor germlines (Angelo and Van Gilst, 2009; Michael-
son et al., 2010; Pinkston et al., 2006). To regulate stem cell pro-
liferation in the Drosophila and C. elegans germline, insulin-like
signaling acts through its canonical pathway, PI3K (AGE-1 in
C. elegans), and the AKT/AKT-1 serine threonine kinase to phos-
phorylate and inactivate the FOXO/DAF-16 forkhead transcrip-
tion factor (Cavaliere et al., 2005; Michaelson et al., 2010).
Thus, in flies and worms, insulin signaling acts as a relay system
cells. No link, however, has been observed between insulin
signaling and meiotic progression.
During oogenesis in mammals and C. elegans, but not in
Drosophila, activation of ERK, the terminal kinase of the
conserved RTK-RAS-ERK signaling pathway, plays a key role
in meiotic maturation (Ivanovska et al., 2004; Lee et al., 2007;
Miller et al., 2001; Verlhac et al., 1993). In mammalian oocytes,
sustained ERK activation for ?12 hr from prometaphase of
meiosis I (MI) until the end of meiosis II, turning off just minutes
before fertilization, is essential for many steps of meiotic pro-
gression, such as spindle migration during MI, the first meiotic
division, prophase progression of meiosis II, arrest at meiosis
II, and the transition from metaphase of MI through metaphase
of meiosis II (Brunet and Maro, 2005; Choi et al., 1996; Verlhac
et al., 1996). During oogenesis in mammals, ERK is activated
by Mos, a meiosis-specific serine-threonine kinase, that takes
Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc. 227
the place of RAF and activates MEK in the canonical ERK
pathway (Roy et al., 1996; Verlhac et al., 1996). During meiosis,
Mos activation appears to be under translational control and
ERK-mediated positive feedback (Charlesworth et al., 2002;
Matten et al., 1996) rather than growth factor signaling. Studies
from Xenopus oocytes, however, implicate progesterone activa-
tion as an upstream signal that activates Mos (Frank-Vaillant
et al., 1999), potentially placing meiotic progression in Xenopus
oocytes under physiological control.
In C. elegans oocytes, sustained activation of ERK also drives
meiotic progression (Lee et al., 2007). Here, ERK is activated by
the conserved RAS-RAF-MEK cascade, starting in the pachy-
tene phase of meiotic prophase I and continuing for ?18 hr
into the diplotene stage of MI (Lee et al., 2007). During this
time, active ERK regulates many events required for meiotic pro-
gression, such as pachytene progression (into diplotene),
plasma membrane organization of pachytene cells, germ cell
apoptosis, and oocyte growth (Arur et al., 2009; Church et al.,
1995; Gumienny et al., 1999; Lee et al., 2007). The upstream
signals that trigger activation of the RAS-ERK pathway during
meiotic progression in worms are unknown.
The C. elegans oogenic germline represents a powerful model
system in which to identify the upstream pathways that activate
ERK during meiosis. Activation of the RAS-ERK pathway occurs
termed zone 1 in this article, MPK-1 (ERK) activation is required,
as noted, for progression of meiotic prophase I; in the proximal
region of the germline, termed zone 2 in this article, MPK-1 acti-
tion of oocytes (Miller et al., 2001). This bimodal activation
pattern of MPK-1 can be directly visualized by the presence of
the activated, diphosphorylated form of MPK-1 (dpMPK-1; Fig-
ure 1A). Prior work identified that a sperm-derived signal acts
through an Ephrin receptor tyrosine kinase (RTK) (Miller et al.,
2001, 2003) to activate MPK-1 in the proximal germline
(zone 2), ensuring that oocytes ovulate only in the presence of
sperm. However, neither the signal nor the receptor that acti-
vates MPK-1 in zone 1 to drive meiotic progression has been
ceptor DAF-2 couples external nutritional conditions to meiotic
progression by activating MPK-1 in zone 1. In the presence of
food, DAF-2 activates MPK-1 in zone 1, promoting meiotic pro-
gression and oocyte production; in the absence of food, DAF-2
does not activate MPK-1 in zone 1, and meiotic progression is
stalled, resulting in loss of oocyte production. In this activity,
DAF-2 acts through the RAS-RAF-MEK cascade rather than
the canonical PI3K/AKT/FOXO pathway. Thus, the C. elegans
germline coordinates two distinct steps of meiosis with distinct
external cues, ensuring that mature gametes are produced in
the presence of both sperm and favorable nutritional conditions.
DAF-2 Activates MPK-1 during Meiotic Prophase I in the
To identify the receptor that activates MPK-1 in zone 1, we first
tested if the epidermal growth factor (EGF) receptor (LET-23)
or fibroblast growth factor (FGF) receptor (EGL-15), canonical
activators of the RAS-ERK pathway in many species, are
required for MPK-1 activation in zone 1. Germlines obtained
from worms homozygous mutant for null alleles of let-23 or
from those in which egl-15 function was depleted specifically
in the germline, via the use of rrf-1 animals (see Supplemental
Experimental Procedures available online), displayed wild-type
dpMPK-1 levels and oocyte development (Figures S1A–S1C).
Thus, we concluded that neither LET-23 nor EGL-15 regulates
MPK-1 in the worm germline.
The RAS-ERK pathway is typically activated by an RTK (Sun-
daram, 2006). The C. elegans genome contains 29 classified and
11 unclassified RTKs (Plowman et al., 1999). Thus, to determine
if an RTK activates MPK-1 in zone 1 of the germline, we per-
formed germline-specific RNAi on the major family member of
the 11 classes of RTKs (Table S1), scoring animals by differential
interference constrast (DIC) imaging for mpk-1-like loss-of-func-
tion germline phenotypes (Lee et al., 2007). Of the 11 RTKs
analyzed, only RNAi of the daf-2 type-1 insulin-like growth factor
receptorelicitedgermline phenotypes indicativeoflossofmpk-1
function (Figures S1D and S1E), suggesting that DAF-2, a type-1
insulin-like receptor, activates MPK-1 in zone 1 of the germline.
During C. elegans development, DAF-2 regulates entry into
and exit from the dauer state, an alternative dormant state that
worms enter into at developmental stage 2 (L2) in response to
stressed environmental conditions (e.g., lack of food) (Evans
DAF-2 signals through AGE-1 (PI3-kinase) and AKT-1 to inhibit
DAF-16 function and bypass entry into dauer. Under stressed
conditions, DAF-2 signaling is inhibited, and active DAF-16 in-
duces worms to enter dauer. Reactivation of DAF-2 signaling is
normally absolutely required for worms to exit dauer and
continue development, although loss of daf-16 function can
both trigger exit from the dauer state and suppress entry into
the dauer state in the absence of daf-2 (or age-1 or akt-1) func-
tion (Dorman et al., 1995; Lin et al., 2001). Thus, loss of daf-2
state; in these worms, the germline, which develops during
young adulthood just after the developmental stage 4 (L4) larval
molt, never forms. Thus, the early requirement for DAF-2 to
bypass or exit the dauer phase may have occluded discovery
of a subsequent role for DAF-2 in the adult germline.
To circumvent this early requirement for daf-2 function, we
used three temperature-sensitive (ts) alleles of daf-2 and as-
ative of loss of mpk-1 function (Table S2). We allowed wild-type
and daf-2 mutant animals to develop at the permissive tempera-
ture (15?C) until young adulthood (mid-L4 stage) and then shifted
the animals to the restrictive temperature of 25?C for 12–24 hr.
This treatment had no effect on wild-type animals: their germ-
lines exhibited wild-type levels of dpMPK-1 and developed the
characteristic linear row of seven to eight oocytes (Figure 1A).
reductionin dpMPK-1levels inzones 1and 2(Figures 1B,1I,and
S1; compare Figures S1E and S1G to Figures S1L–S1O) and
multiple phenotypes indicative of reduced mpk-1 function (Table
S2) (Lee et al., 2007): the presence of a few large, disorganized
oocytes (Figure 1B and Figure 1E), defects in meiotic prophase
I progression (Figure 1D), and increased germ cell apoptosis
(Figure S1G). More complete elimination of daf-2 function either
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
228 Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc.
by Brisbin et al. (2009); a third is that DAF-18 regulates RAS-
MPK-1 activity downstream to DAF-2 independent of its phos-
phatase activity. Consistent with the last idea, PTEN has been
found to inhibit the function of other proteins that promote
growth via protein-protein interactions rather than through its
phosphatase action (Okumura et al., 2005; Song et al., 2011).
Clarifying the molecular basis through which DAF-18 regulates
DAF-2 signaling in the C. elegans germline independently of
PI3K thus represents a key area of future study.
The Caenorhabditis elegans Germline Couples Oocyte
Production and Maturation to Two Distinct External
Our work, together with that of Miller et al. (2001), indicates that
the C. elegans germline couples two distinct external cues to
meiotic progression and oocyte maturation and ovulation, in
both cases, through the activation of the ERK signaling
pathway. Here, we showed that, in the presence of food,
DAF-2 functions in zone 1 to activate the RAS-MPK-1 pathway
and drive meiotic progression. Previously, Miller et al. (2001)
showed that the presence of sperm activates MPK-1 in zone
2 and drives oocyte maturation, ovulation, and fertilization. Inte-
gration of these mechanisms generates a seemingly adaptive
system that ensures that organismal resources are shepherded
toward procreation only under conditions in which fertilization
can occur and that favor survival of the progeny (Figure 8).
Thus, in the presence of favorable nutrient conditions and
sperm, the C. elegans germline continually produces oocytes,
which are then fertilized, and the resulting progeny is born un-
der hospitable conditions. In the absence of either signal,
oogenesis is inhibited in mechanistically distinct ways: in the
absence of food and daf-2 signaling, MPK-1 activation does
not occur in zone 1 and meiotic progression is stalled (over
time, dpMPK-1 is also lost in zone 2); in the absence of sperm
results, MPK-1 activation in zone 2 does not occur (over time,
dpMPK-1 is also lost in zone 1 but not before a stockpile of
oocytes has been produced). In either scenario, the germline
is poised to respond to a change in environment—the appear-
ance of food or sperm—via rapid reactivation of MPK-1 in zone
1 (food) or zone 2 (sperm) and oocyte production, maturation,
and fertilization. Thus, C. elegans oogenesis appears to provide
an elegant example of how evolution has sculpted an adaptive
organ system that helps ensure the survival of its (fittest)
Indicated genotypes were grown on nematode growth medium (NGM) plates
with E. coli OP50 bacteria to the indicated developmental stage and then
transferred to an unseeded NGM plate (minus peptone) as described earlier
and starved for indicated time points (Angelo and Van Gilst, 2009).
NGM plates minus cholesterol were supplemented with 1 mM DA (Sharma
et al., 2009) and seeded with E. coli OP50 for use.
Dissections and Staining
Dissections were performed as described earlier (Arur et al., 2009). All dissec-
tions were performed under 5 min (immediately after adding levamisole) to
achieve optimal dpMPK-1 staining. The dissected germlines were fixed in
3% paraformaldehyde for 10 min, followed by a postfix in 100% methanol at
?20?C. The fixed germlines were then processed for immunofluorescence
staining as described elsewhere (Arur et al., 2009).
Supplemental information includes Supplemental Experimental Procedures,
five figures, and two tables and can be found with this article online at http://
A.L.L., J.C., and S.A. conceived and designed the study. A.L.L., J.C., H.-J.J.,
M.D., M.S., C.K., and S.A. performed experiments and analyzed data. S.A.
wrote the paper.
Worm strains were obtained from the C. elegans Genetics stock center at the
University of Minnesota, funded by the National Institutes of Health (NIH) (P40
OD010440). We thank Jim Skeath, Awdhesh Kalia, and Jill Schumacher for
their critical review of the manuscript and valuable discussions and David
Mangelsdorf for providing DA and the idea to test daf-2 mutant worms on
DA to assay adult germline phenotypes. A grant from the Cancer Prevention
and Research Institute of Texas (RP101502 to J.C.), an NIH grant
(GM98200), and an M.D. Anderson Cancer Center Support grant from the Na-
tional Cancer Institute to S.A. supported this work.
Received: December 29, 2012
Revised: July 2, 2013
Accepted: September 12, 2013
Published: October 10, 2013
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