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.
Figure 1. daf-2 Regulates MPK-1 Activation and Function in Zone 1
Dissected C. elegans hermaphrodite germlines, oriented from left (mitotic cells, indicated by an asterisk) to right (oocytes), and stained for membrane (green),
dpMPK-1 (red), or DNA (DAPI, white).
(A) Wild-type germlines (20 hr/L4 at 25?C) exhibit dpMPK-1 in zones 1 and 2 and linear formation of oocytes (marked ?1, ?2, and ?3 per birth order). sp, sperm.
(B) daf-2 mutant germlines exhibit reduced dpMPK-1 in zones 1 and 2 and large, disorganized oocytes.
(C and D) Germlines from wild-type (C) and daf-2 mutant worms (D) stained for DNA (white) and lamin (green). daf-2 mutant germlines exhibit delayed pachytene
progression (PP). Wild-type germlines display normal PP and oocyte formation (arrowheads).
(E) DIC image of whole mount wild-type and daf-2 mutant germlines. In daf-2 loss-of-function animals, oocytes are reduced in number with a large ?1 oocyte.
(F and G) Wild-type and daf-2 mutant germlines maintained at 25?C for 8 hr (at 20 hr/L4) stained with dpMPK-1 and lamin. daf-2 mutant germlines display a
(legend continued on next page)
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc. 229
via germline-specific daf-2 RNAi or through the use of dafach-
ronic acid (DA) to bypass dauer formation in daf-2 loss-of-func-
tion animals (Experimental Procedures) led to a complete loss of
detectable MPK-1 activation and germline phenotypes essen-
in the germline (Figures S1E–S1G). These data suggest that
DAF-2 regulates MPK-1 in the germline and that its loss leads
to germline phenotypes that mirror those elicited upon abroga-
tion of MPK-1 function.
TheC.elegansgermline develops inanassembly-line manner,
with germ cells developing into mature oocytes (zone 2) within
3–4 hr after they exit the pachytene phase of meiotic prophase
I (zone 1) (Lee et al., 2007). Thus, the observed loss of MPK-1
activity in zones 1 and 2 of daf-2 mutant germlines could arise
because DAF-2 regulates MPK-1 in both zones or because
DAF-2 regulates MPK-1 specifically in zone 1, and loss of
MPK-1 function in zone 1 leads to loss of MPK-1 activation in
zone 2 due to subsequent defects in oocyte development. To
distinguish between these models, we maintained daf-2 mutant
animals at the permissive temperature of 15?C for 12 hr past L4,
shifted them to the restrictive temperature of 25?C for 8 hr, and
then assayed the resulting effect on dpMPK-1 levels in zones 1
and 2. daf-2 mutant germlines that underwent this treatment
exhibited a specific loss of dpMPK-1 in zone 1 (pachytene),
with little or no effect on dpMPK-1 levels in zone 2 (proximal
oocytes) (Figures 1F and 1G). Quantification of the decrease in
dpMPK-1 levels in zone 1 from daf-2 mutant germlines relative
to wild-type revealed a 90% reduction of dpMPK-1 levels in
zone 1 but a <1% change in zone 2 (Figures 1H, 1I, and S1H–
S1O). In addition, daf-2 mutant germlines contained two to three
oocytes versus seven to eight in wild-type, suggesting that the
reduction in daf-2 function in zone 1 resulted in the halting of
support the model that DAF-2 regulates MPK-1 activation in
zone 1 of the germline and, through this regulation, triggers pro-
gression of MI.
DAF-2 Couples the Presence of Nutrition to RAS-MPK-1
Pathway Activation Independently of the AGE-1/AKT-1/
The insulin pathway is known to couple nutrient status to cell
and organismal growth: in the presence of food, insulin signaling
is active and promotes growth; in the absence of food, insulin
signaling is inactive and growth is inhibited (Neufeld, 2003). To
test whether DAF-2 couples nutritional status to activate the
RAS-MPK-1 pathway and oogenesis in C. elegans, we starved
fully developed wild-type worms and assayed the resulting ef-
fect on dpMPK-1 in zone 1 and oocyte development. Prolonged
starvation exhibits pleiotropic effects on germ cell development
independent of any one signaling pathway (Angelo and Van
Gilst, 2009). Thus, we starved animals for varying times to test
whether loss of nutrition specifically affects dpMPK-1 prior to
manifestation of any visible effects of starvation. We starved an-
imals for 30 min, 1 hr, 2 hr, 4 hr, 6 hr, and 24 hr past the L4 stage
of development (Figure S2; data not shown) and then assayed
germlines for dpMPK-1. Starvation for as little as 1 hr reduced
dpMPK-1 levels in zone 1 (Figure S2B), and a 2 hr starvation re-
sulted in near-complete loss of dpMPK-1 levels in zone 1 but not
in zone 2 (Figure 2C, compared to Figure 2A and Figure S2).
Starvation for 4 and 6 hr resulted in defects in pachytene pro-
gression and the formation of large, disorganized oocytes,
with very reduced MPK-1 activation in zone 2 (Figures S2C
and S2D), similar to mpk-1 loss in the germline (Arur et al.,
2009). Quantification of the decrease in dpMPK-1 levels in 2-
hr-starved animals relative to fed animals revealed a >90%
reduction in dpMPK-1 levels in zone 1 but little or no change
in dpMPK-1 levels in zone 2 (Figures 2C and 2D). Thus, transient
starvation of adult worms yields the same germline phenotype
as transient reduction of daf-2 function: a specific reduction of
MPK-1 activation during pachytene progression in zone 1 and
the formation of large disorganized oocytes (compare Figure 1B
to Figure 1A, and compare Figure 1H to Figure 1I; Figures 2C
The effect of starvation is reversible: animals starved for 2 hr
and then re-fed for 4 hr displayed normal levels of dpMPK-1
and reinitiated oocyte production (Figure 2E). In addition, starva-
tion of daf-2(e1370) mutant animals for 2 or 4 hr at the restrictive
temperature did not exacerbate the loss of dpMPK-1 or the
oocyte phenotype (Figures 2G–2J), suggesting that nutrition
and daf-2 act in the same pathway to regulate MPK-1 activation
and oocyte production. Thus, DAF-2 appears to couple nutri-
tional status to the activation of MPK-1 during meiotic prophase
and, thus, oocyte generation.
Caloric restriction has been shown to lengthen C. elegans
lifespan (Lakowski and Hekimi, 1998). Mutations in genes that
disrupt pharyngeal function and reduce normal feeding (‘‘eat’’
mutations) result in calorically restricted animals that live
longer. With respect to aging, this caloric restriction pathway
functions in parallel to the insulin signaling pathway (Lakowski
and Hekimi, 1998). We tested whether caloric restriction had
an impact on dpMPK-1 in zone 1 and oocyte production by
analyzing two distinct eat-2 mutant germlines and found that,
even though the animals were somatically slow growing and
scrawny in appearance, loss of eat-2 function had no impact
on zone 1 MPK-1 activation or oocyte production (Figures
S2E–S2G). Thus, as observed for aging, insulin signaling acts
independently of the caloric restriction pathway to regulate
DAF-2 acts through the AGE-1/AKT-1 cascade to inhibit DAF-
16 functionto regulate dauer formation, aging, and germline pro-
liferation in C. elegans (Michaelson et al., 2010). We thus asked
whether DAF-2 acts through age-1 and akt-1 to regulate MPK-
1 activation in zone 1. Since AGE-1 and AKT-1, like DAF-2, regu-
late entry into the dauer state in C. elegans, we performed RNAi
analysis of age-1 and akt-1 in wild-type animals and in rrf-1 ani-
wild-type germlines, the dpMPK-1 staining in somatic sheath cell is masked by intense dpMPK-1 accumulation in germ cells. Experiments were performed five
times; 25–30 germlines were analyzed each time.
(H and I) ImageJ-based pixel intensity of dpMPK-1 in zone 1 from wild-type and daf-2 mutant germlines depicted in (F) and (G). The x axis represents position
along the germline; the y axis represents dpMPK-1 pixel intensity. Scale bar, 20 mm.
See also Figure S1 and Tables S1 and S2.
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
230 Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc.
akt-1 in wild-type worms triggered entry into the dauer state
germlines with loss of akt-1 or age-1 exhibit normal dpMPK-1 in
zone 1 and oocyte development (Figures S3A–S3D). Thus,
DAF-2 does not appear to function through the AGE-1 or
AKT-1 to regulate dpMPK-1 in zone 1.
Figure 2. daf-2 Couples Nutritional Cues to MPK-1 Activation in Zone 1
(A–J) (A), (C), (E), (G), and (I) show dissected adult (24 hr past L4) hermaphrodite germlines stained for DNA (DAPI, white) and dpMPK-1 (red). In (A), wild-type
germlines from fed conditions exhibit two zones of MPK-1 activation and six to seven oocytes. In (C), wild-type germlines from starved (2 hr) conditions exhibit
reduced dpMPK-1 in zone 1 but not zone 2. In (E), wild-type germlines from animals re-fed upon starvation reveal restoration of dpMPK-1 in zone 1. In (G) and (I),
daf-2 mutant germlines exhibit reduced dpMPK-1, PP defects, and one large oocyte both on and off food. (B), (D), (F), (H), and (J) show quantitative measures of
dpMPK-1 levels from (A), (C), (E), (G), and (I) taken with ImageJ. The x axis depicts germ cell position along the length of the germline, and the y axis measures the
dpMPK-1 accumulation as pixel intensity. The experiment was performed four times; 50 germlines were analyzed each time. Scale bar, 20 mm.
See also Figure S2.
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc. 231
Next, we asked whether daf-2 acts through daf-16 to activate
MPK-1 in zone 1. In the canonical insulin signaling pathway,
daf-2 activates insulin signaling by repressing daf-16, and loss
of daf-16 results in active insulin signaling regardless of whether
daf-2 function is present (Apfeld and Kenyon, 1998). Thus, if
daf-2 acts through daf-16 to activate MPK-1 in zone 1 of the
germline, loss of daf-16 should reverse the effects of both star-
vation and loss of daf-2 function on dpMPK-1 in the germline.
However, we found that daf-16 null mutant worms starved for
2hrbehavedidentically to wild-typeworms: theydownregulated
Thus, loss of daf-16 function fails to reverse the effects of starva-
tion, consistent with daf-2 acting independently of daf-16 to acti-
vate MPK-1 in zone 1 of the germline.
Next, we assayed a daf-16::GFP transgene, muIs61, that fully
rescues the daf-16(mu86) null mutant background to follow
line itself, DAF-16::GFP is barely detectable, indicating that
daf-16 is expressed at low levels in this tissue. DAF-16::GFP,
however, is expressed at high levels in the somatic gonadal
sheath cells, localizing to the nuclei of these cells under normal
fed conditions (Figure 3C). As DAF-2 signaling leads to the phos-
phorylation of DAF-16 and its subsequent nuclear exclusion, our
observation suggests that DAF-16 acts independently of DAF-2
in the somatic gonad, a tissue that exerts profound nonautono-
mous control over many aspects of germline development,
including mitosis, meiotic progression, and ovulation (McCarter
et al., 1997).
Due to DAF-16 expression in the somatic gonad, we assessed
the effect of removing daf-16 function from daf-2 mutant worms
via germline-specific depletion of daf-16 function (daf-16 RNAi in
the rrf-1 background) and systemic depletion of daf-16 function
via RNAi or the use of daf-16 null alleles. Depletion of daf-16
function in rrf-1;daf-2(e1370) mutant worms suppressed the
daf-2 proliferation phenotype observed in the mitotic zone of
the germline (as previously reported by Michaelson et al.,
2010; compare Figure 3D to Figure 3E) but had no effect on
the loss of dpMPK-1 in zone 1 or decreased oocyte production
Figure 3. daf-16 Does Not Function Downstream to daf-2/Nutritional Cues to Regulate Zone 1 MPK-1 Activation and Oocyte Development
Dissected adult hermaphrodite germlines stained for DNA (DAPI, white), dpMPK-1 (red), or REC-8 (green).
(A and B) daf-16(mu86) germlines on either fed (A) or starved (B) conditions. daf-16 germlines from fed animals reveal normal dpMPK-1 in zone 1 and oocyte
development (A) but reduced dpMPK-1in zone 1 and decreased oocyte production in the starved condition(B). RNAi analysis wasperformed in triplicate, and 50
germlines were analyzed for each genotype. The starvation experiment was performed five times, and 20–25 germlines were analyzed each time.
(C) Whole-mount DIC and GFP analysis of muIs61, DAF-16::GFP animals. Arrows indicate nuclear staining of DAF-16::GFP in somatic gonadal sheath cells.
(D and E) Germlines from rrf-1;daf-2 animals at the restrictive temperature with gfp (D) or daf-16 (E) RNAi treatment. In (E), reduction of daf-16 in daf-2 mutant
animals resultsinrestoration ofthemitotic proliferativegermcells(greenarrow)butdoes notrescuePPdefects,stalledoocytedevelopment,orMPK-1activation
in zone 1.
(F) Germlines from daf-16;daf-2 double mutant animals have normal mitotic zone development and dpMPK-1. ++. nonspecific signal from the intestine. Scale
bar, 20 mm.
See also Figure S3.
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
232 Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc.
observed in the proximal germline of daf-2 mutant worms
(compare Figure 3D to Figure 3E). This result indicates that,
within the germline, daf-2 does not signal through daf-16 to acti-
vate MPK-1. In contrast, systemic loss of daf-16 function in a
daf-2 mutant background, via the use of either RNAi or three
daf-16 null alleles (see Experimental Procedures), rescued both
the germline proliferation phenotype and the loss of dpMPK-1
in zone 1 (Figure 3F). This result indicates that systemic loss of
daf-16 impacts germline development, and MPK-1 activation
within it, in an indirect manner via a function in the somatic
gonadal sheath cells. We believe that the simplest interpretation
for all of our results integrated together is that, within the germ-
line, daf-2 acts independently of age-1, akt-1, and daf-16 to acti-
Figure 4. MPK-1 and MEK-2 Are Epistatic to
Dissected adult hermaphrodite germlines stained
with dpMPK-1 (red) and DNA (DAPI, white).
(A) Germlines obtained from GFP::DAF-2 trans-
through zone 1 (yellow line).
(B) Germlines from vizIs23 (GFP::DAF-2);daf-
2(e1370) animals at 25?C exhibit normal dpMPK-1
levels in zone 1 and oocyte development.
(C and D) Germlines obtained from DAF-2 over-
expression animals upon RNAi treatment with
mpk-1 or mek-2 reveal mpk-1 loss-of-function
phenotypes. The RNAi experiment was performed
three times; 30–35 germlines were assayed each
time. Scale bar, 20 mm.
See also Figure S4.
vate MPK-1 and meiotic progression and
that a daf-2-independent role for DAF-16
in somatic gonadal sheath cells accounts
for the restoration of dpMPK-1 levels in
zone 1 in daf-16, daf-2 double mutant
DAF-2 Signals through the RAS-
MPK-1 Cascade to Regulate
Meiotic Progression and Oocyte
Our data indicate that DAF-2 activates
(zone 1). Prior work indicates that the
RAS (LET-60), RAF (LIN-45), and MEK
(MEK-2) module activates MPK-1 during
meiotic prophase I in the C. elegans
germline (Lee et al., 2007). Thus, we
asked if DAF-2 signals via this cascade
to activate MPK-1 and regulate germline
development. To assay if DAF-2 acts
through MEK-2 and MPK-1, we gener-
ated a GFP::DAF-2 transgene (Figure 4A),
wherein GFP::DAF-2 expression was
placed under the control of the germ-
line-specific pie-1 promoter (Supple-
mental Experimental Procedures). We
first tested whether the presence of the
transgene rescues the daf-2(e1370) mutant phenotype at the
restrictive temperature and found that it restored normal oocyte
development and zone 1 MPK-1 activation (Figure 4B). Analysis
of the localization of GFP::DAF-2 in the germline reveals that
GFP::DAF-2 localizes to the cell membrane, the cytoplasm,
and cytoplasmic vesicles (Figure S4B, inset).
sulted in a heightened accumulation of dpMPK-1 in zone 1 and
the loop region (Figure 4A), consistent with DAF-2 activating
MPK-1 in zone 1. Depletion of either mek-2 or mpk-1 function
in this background resulted in complete loss of dpMPK-1 levels
in meiotic prophase I and the loop region and also produced
phenotypes similar to mek-2 and mpk-1 mutants: pachytene
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc. 233
arrest of cells and clumping and disorganization of pachytene
cells, as evidenced by the ‘‘holes’’ in zone 1 (Figures 4C and
4D). Thus, daf-2 requires mek-2 and mpk-1 for its function in
zone 1, suggesting that DAF-2 acts upstream of MEK-2 and
MPK-1 to regulate oogenesis in the germline.
In support of daf-2 acting through the RAS-MPK-1 pathway in
epistatic to mutations in daf-18. DAF-18, the worm PTEN homo-
log, negatively regulates DAF-2 signaling downstream of recep-
display heightened dpMPK-1 levels in zone 1 and an increased
ovulation rate (Figure 5B compared to Figure 5A), presumably
due to both increased insulin signaling and a reported role for
DAF-18 downstream to VAB-1 Eph receptor signaling to nega-
tively regulate oocyte ovulation (Brisbin et al., 2009). To test
whether mpk-1 functions downstream to daf-18 during zone 1
activation and meiotic progression, we generated daf-18;mpk-
1 double mutants and conducted RNAi analysis of mpk-1 in rrf-
1;daf-18 animals. Worms of both genetic backgrounds exhibited
germline phenotypes indistinguishable from mpk-1 mutant
worms: pachytene progression defects, increased germ cell
death, and loss of oocyte production (Figure 5C; data not
shown). These data suggest that DAF-18 attenuates DAF-2-
mediated activation of the RAS-MPK-1 pathway in zone 1 of
Consistent with loss of daf-18 function leading to increased
daf-2 signaling in zone 1, the germlines of daf-18 mutant are
partially resistant to starvation. Germlines of daf-18(ok480)
worms subjected to a 2-hr starvation retained elevated
dpMPK-1 levels in zone 1 and continued to produce oocytes,
albeit at a decreased rate relative to fed daf-18 mutant worms
(Figure 5D). Germlines of daf-18(ok480) animals subjected to a
4-hr starvation, however, exhibited a starvation phenotype:
and contained only one to two large oocytes as in wild-type
animals (Figure 5E). Thus, daf-18 mutant animals are resistant
to short, but not extended, stretches of starvation, consistent
with the ability of DAF-18 to oppose insulin signaling down-
stream of DAF-2 receptor activation.
If nutrition and daf-2 act through the RAS-MPK-1 pathway to
regulate oogenesis, then constitutive activation of let-60 (ras)
should reverse the effect of loss of daf-2 function and starvation
with respect to oocyte development and production. To test this
model, we used the gain-of-function, temperature-sensitive
let-60(ga89 gf) mutant. At the permissive temperature (20?C),
let-60(ga89 gf) animals exhibit normal germline development
but heightened dpMPK-1 levels in zone 1 and the loop region
(Figure 6A). At the restrictive temperature (25?C), the germlines
of these animals exhibit hyperactivation of dpMPK-1 and strong
gain-of-function phenotypes, including the production of multi-
ple,smalloocytes(Figure6C)(Lee etal.,2007).At thepermissive
temperature, we found that the germlines of let-60(ga89 gf) ani-
mals were partially resistant to starvation: dpMPK-1 was down-
regulated after 4 hr of starvation but not after 2 hr of starvation
(Figure 6B). At the restrictive temperature, however, the germ-
lines of daf-2(e1370); let-60(ga89 gf) (Figure 6F) or let-60(ga89
gf) animals starved for 2 (Figure 6D) or 4 hr (data not shown)
the germlines retained elevated dpMPK-1 levels in zone 1 and
Figure 5. daf-18/PTEN Functions Downstream to Nutritional Cues
and Upstream to mpk-1
Dissected adult hermaphrodite germlines stained for DNA (DAPI, white) and
active MPK-1 (red).
(A and B) Germlines from wild-type (A) or daf-18(ok480) (B) animals. Loss of
daf-18 results in continuous dpMPK-1 through zone 1 (yellow line) and
endomitotic oocytes in the germline (arrows) and the uterus.
(C) daf-18;mpk-1 double mutants exhibit pachytene arrest and no oocyte
production, as shown in (B).
(D and E) daf-18 loss-of-function animals after 2 hr (D) and 4 hr (E) of starvation
exhibit downregulation of dpMPK-1 in zone 1 and suppression of the hyper-
ovulation phenotype (D). Starvation for 4 hr (E) results in further reduction in
dpMPK-1 and oocyte disorganization phenotypes (arrowheads). Experiments
were performed three times; 50–60 germlines were analyzed each time. Scale
bar, 20 mm.
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
234 Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc.
continued to produce small oocytes. This was true, even though,
after the 4-hour starvation, let-60 animals were overtly thinner
(and thus starved of nutrients). Thus, the let-60 gain-of-function
phenotype is epistatic to daf-2 and starvation, supporting the
idea that nutrition signals via DAF-2 and the RAS-MPK-1
pathway to activate MPK-1 in zone 1 and promote progression
To assay whether overexpression of the DAF-2 receptor was
also epistatic to starvation, we starved wild-type worms that
harbored the GFP::DAF-2 transgene for 2, 4, and 6 hr. These
worms displayed partial resistance to starvation effects after 2
and 4 hr (Figures 6D–6L), but not 6 hr (Figure 6L), suggesting
that the receptor may be downregulated or turned over in the
absence of signal over time.
DAF-2 Acts in a Homeostatic Regulatory Mechanism
that Couples Oogenesis to Environmental Conditions
In wild-type hermaphrodites, two independent signals activate
MPK-1 in the germline: daf-2 and nutrition in zone 1; and the
sperm signal in zone 2 (Miller et al., 2001). To isolate the effect
of DAF-2 and nutrition-mediated activation on MPK-1 in zone 1
and germline development from sperm-mediated activation of
Figure 6. LET-60 RAS Functions Downstream to DAF-2 and Nutritional Cues to Regulate MPK-1 Activation
Dissected adult hermaphrodite germlines stained for DNA (DAPI, white) and dpMPK-1 (red).
(A–F) let-60(ga89 gf) animals in (A) through (D) and daf-2;let-60(ga89 gf) animals in (E) and (F) were fed or starved for 2 hr. In (A) through (D), at 25?C on starvation
(D),let-60(ga89gf)wormsdisplaycontinuousdpMPK-1throughzone1(yellowline)andproduce doublerowsofsmalloocytes (arrowheads). At20?C,let-60(ga89
gf) worms downregulate dpMPK-1 in zone 1 (B). In (E) through (F), daf-2;let-60(ga89 gf) worms display continuous dpMPK-1 and multiple oocytes both on and off
food at 25?C. Experiments were performed three times; each time, 40 germlines were analyzed.
(G–L) Quantitative measures of dpMPK-1 levels in zones 1 and 2 from germlines of fed or starved GFP::DAF-2 (vizIs23) worms, taken with ImageJ. The x axis
depicts germ cell position along the length of the germline; the y axis measures the dpMPK-1 levels as pixel intensity. Scale bar, 20 mm.
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc. 235
germlines of fem-3 animals are essentially identical to wild-type
hermaphrodites (Figures 7A and 7C), except that they lack
sperm and, thus, sperm-mediated activation of MPK-1 in zone
2(Figures 7D and7F). Young females (6hrafterL4 molt) possess
relatively normal levels of dpMPK-1 in zone 1 compared to age-
matched hermaphrodites (Figure 7A; compare Figure 7C to
Figures 7D and 7F). Interestingly, in the absence of sperm-
dependent high zone 2 MPK-1 activation, a dpMPK-1 signal in
pendent signal (Figure 7). Much like the hermaphrodites, fem-3
germlines generate five to six oocytes, but in contrast to the her-
maphrodites, these oocytes arrest in the absence of sperm and
there is no dpMPK-1 in zone 2 (compare Figure 7A to Figure 7D;
compare Figure 7C to Figure 7F). Upon introduction of sperm,
these arrested oocytes activate MPK-1 in zone 2 and undergo
maturation, ovulation, and fertilization (Miller et al., 2001).
To investigate the effect of loss of daf-2 function or starvation
specifically on zone 1 MPK-1 activation, we analyzed the germ-
lines of young daf-2; fem-3 double mutants or of fem-3 animals
starved for 2 hr. In both cases, we observed the same effect:
the germlines displayed a dramatic loss of dpMPK-1 in zone 1
and mpk-1 like loss-of-function phenotypes, such as loss of
pachytene cell membranes, pachytene progression defects,
and the formation of large, disorganized oocytes (compare Fig-
ure 7H to Figure 7E and Figure 7I to Figure 7F)—phenotypes
essentially identical to those observed in daf-2 hermaphrodites
or starved wild-type animals (compare Figures 1B–1E to Fig-
ure 2C and Figure S1). Thus, as observed in hermaphrodites,
DAF-2 acts through MPK-1 to drive the formation of oocytes in
Figure 7. daf-2 and Nutritional Cues Regu-
late dpMPK-1 in Zone 1 for Oocyte Produc-
(A–K) Dissected hermaphrodite germlines, in (A)
and (B), and female germlines, in (D), (E), (G), (H),
(J), and (K), stained for DNA (DAPI, white) and
dpMPK-1 (red). In (A) and (B), young (6 hr/L4)
fem-3 heterozygous germlines on food (A) or
starved (B) for 2 hr are shown. Germlines from fed
worms exhibit dpMPK-1 in zones 1 and 2 and
three to four oocytes. Starved germlines display
reduced dpMPK-1 in zone 1, defects in pachytene
progression, and large oocytes (blue bracket). (C)
shows a quantitative measure of dpMPK-1 pixel
intensity in zones 1 and 2 from (A) and (B) was
position along the length of the germline, and the y
axismeasuresthedpMPK-1 accumulation aspixel
intensity. In (D) and (E), young female germlines
from fed or starved (2 hr) conditions are shown.
Fed females display normal dpMPK-1 in zone 1.
zone 1, pachytene progression defects, oocyte
loss, and a larger ?1 oocyte (blue bracket). (F)
shows a quantitative analysis of dpMPK-1 levels
from (D) and (E), acquired using ImageJ, displayed
as per (C). (G) and (H) show young daf-2 loss-of-
function females at 15?C (G) or 25?C (H). Young
daf-2 females at 25?C display defects in pachy-
tene progression, exhibit a reduction in oocyte
number, and contain large oocytes (blue bracket).
(I) shows a quantitative analysis of dpMPK-1 levels
in germlines analyzed in (G) and (H), displayed as
per (C). (J) and (K) show young let-60(ga89 gf) fe-
males either fed (J) or starved (K).
(L) Quantitative analysis of dpMPK-1 from (J) and
(K), displayed as per (C). Fed let-60(ga89 gf) fe-
males reveal continuous dpMPK-1 in zone 1 and
sperm-independent oocyteactivation.Starved let-
60(ga89 gf) females reveal continuous dpMPK-1
through zone 1 and sperm-independent region in
the oocytes. Experiments were performed five
times; each time, 30–35 germlines analyzed. Scale
bar, 20 mm.
See also Figure S5.
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
236 Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc.
young female worms. Also as observed in hermaphrodites,
constitutive activation of the RAS-MPK-1 pathway, through the
use of the let-60 (ras) gain-of-function allele, is sufficient to drive
oogenesis in young females even when starved. Young, fed or
starved, fem-3(0);let-60(ga89 gf) animals exhibited the same
phenotype: high levels of dpMPK-1 in zone 1, sperm-inde-
pendent zone 2 dpMPK-1, and the continual production of
many small oocytes—phenotypes indistinguishable from those
observed in animals singly mutant for let-60(ga89 gf) (compare
Figures 7J–7L to Figures 6C and 6D). Thus, constitutive activa-
tion of the RAS-MPK-1 pathway in zone 1 is sufficient to drive
oogenesis in the absence of nutrition and sperm signal in young
In contrast to young females, old females (24 hr after L4 larval
molt) possess low levels of dpMPK-1 in zone 1 and a stockpile of
14–16 arrested oocytes (Figure S5C). In these females, starva-
tion did not appreciably reduce dpMPK-1 levels or impact germ-
line morphology beyond that typically observed in old females
that were fed (Figures S5C and S5D). However, when allowed
to mate with males, females within 2 hr old exhibited high levels
underwent maturation, ovulation, and subsequent fertilization
(Miller et al., 2001; data not shown). It is formally possible that
the introduction of sperm, as well as the activation of the sperm
signal, directly activates MPK-1 in zones 2 and 1, but we favor a
different model. We propose that, in the absence of sperm, the
stockpiling of arrested oocytes in old females eventually triggers
production in zone 1. Release of the block would require intro-
duction of sperm and the subsequent resumption of oocyte
maturation and fertilization. Regardless of the exact cause of
this observation, we note that extended loss of MPK-1 activation
in either zone 1 (lack of food) or zone 2 (lack of sperm) ultimately
results in loss of MPK-1 activation in both zones 1 and 2 and
cessation of oocyte production.
‘‘It is not the strongest of the species that survives, nor the most
intelligent that survives. It is the one that is the most adaptable to
change.’’ —Charles Darwin (Darwin, 1859)
Our work suggests the presence in C. elegans of a physiolog-
ical relay system that couples nutrient availability to meiotic
progression during oogenesis through the action of the DAF-2
insulin-like receptor and the RAS-MPK-1 pathway. Below, we
discussthe role of insulin signaling in coupling animal physiology
and development to environmental conditions, the apparent
PI3K-independent function of PTEN during meiotic progression
in the C. elegans germline, and the C. elegans germline as an
organ that coordinates oocyte (and progeny) production to two
independent external cues.
DAF-2 Couples Nutrient Availability to Meiotic
Progression by Activating the RAS-MPK-1 Signaling
The insulin-like signaling pathway has been shown to link meta-
bolic inputs to developmental and cellular outcomes in multiple
different model systems and in humans (Colombani et al.,
2003; Hietakangas and Cohen, 2009; Michaelson et al., 2010).
In Drosophila, the cellular and molecular pathways through
which the insulin-like signaling pathway couples nutritional sta-
tus to cell division, cell growth, and tissue development has
been particularly well delineated. Here, the presence of nutrition
in the form of amino acids has been shown to elicit the fat body,
the fly adipose tissue, to secrete a diffusible signal that triggers
the secretion of insulin-like peptides (ILPs) from a small set of
neurons in the brain (Colombani et al., 2003). The ILPs then
diffuse systemically and activate the insulin-like signaling
pathway in diverse tissues. In the ovary, the insulin-signaling
pathway acts through the canonical PI3K/AKT cascade to regu-
late stem cell proliferation and the uptake of yolk proteins by
maturing oocytes (LaFever and Drummond-Barbosa, 2005).
We suspect that a similar physiological relay system occurs in
C. elegans. In the meiotic germline, we find that the DAF-2 insu-
lin-like signaling pathway responds to the presence of food by
driving progression of MI and oocyte production (Figures 1B–
1E). Lack of either DAF-2 function or nutrition stalls oocyte pro-
meiotic progression via sustained activation of MPK-1 for ?18 hr
in each germ cell, with MPK-1 activity then orchestrating a suite
of biological events that drives meiotic progression and oogen-
esis (Arur et al., 2009; Lee et al., 2007).
DAF-2 activity couples external cues to meiotic progression,
but what ligands activate DAF-2 in zone 1 of the germline, and
what is their cellular source? Several studies suggest that sen-
sory neurons secrete ILPs in response to nutrient availability
during larval development (Apfeld and Kenyon, 1999; Bargmann
and Horvitz, 1991; Michaelson et al., 2010). By analogy, we
speculate that, in adult animals, the presence of food triggers
neurons to secrete ILPs that then act remotely to trigger DAF-
2 receptor activation in zone 1 of the germline. The signals
that trigger ILP secretion in this system, however, remain un-
known. Interestingly, and unlike in the Drosophila ovary, in the
relay system analyzed in this study, the DAF-2 insulin-like
signaling pathway does not utilize the canonical AGE-1/AKT-1/
DAF-16 module and, instead, appears to integrate directly with
the RAS-RAF-MEK-ERK cascade to mediate meiotic prophase
progression and oocyte production. Future work is required to
delineate all of the players—both molecules and tissues—in
this relay system in C. elegans and to reveal the similarities
and differences between the processes in C. elegans and mam-
mals that couple nutrient availability to ERK activation and
Our data also identified a likely DAF-2 independent function of
DAF-16 in somatic gonadal sheath cells to regulate germline
development. DAF-16 is expressed at low levels in the germline
(Michaelson et al., 2010) but at high levels in somatic gonadal
sheath cells (Figure 3C). Removal of daf-16 function specifically
levels in zone 1. However, complete, systemic loss of daf-16
function in daf-2 mutant worms reversed the loss of dpMPK-1
staining in zone 1. As daf-16 is expressed in the somatic gonad
and the somatic gonad exerts significant influence over germline
development (McCarter et al., 1997), we speculate that daf-16
acts in an autonomous manner to regulate the function of
somatic gonadal sheath cells and through them acts in a non-
autonomous manner to influence germline development. In this
function, daf-16 likely acts independently of daf-2, as under
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc. 237
normal fed conditions in which daf-2 signaling is active, DAF-16
localizes to the nucleus of somatic gonadal sheath cells and is,
thus, presumably active (Figure 3C). Recent evidence reveals
that DAF-16 also acts independently of daf-2 via miRNA-medi-
ated regulation of akt-1 in the somatic gonad to promote
longevity in C. elegans (Shen et al., 2012). Thus, in the future, it
will be important to dissect the function of daf-16 in the somatic
gonad and to investigate how it impacts germline development
in a daf-2 and insulin signaling independent manner.
DAF-18/PTEN Acts Independently of PI3 Kinase to
Control Meiotic Progression Downstream to DAF-2
During meiotic progression in zone 1, we find that DAF-2 acts
independently of the canonical AGE-1, AKT-1, and DAF-16
pathway (Figures 3 and S3), and instead functions via the RAS-
RAF-MEK-ERK cascade (Figures 2 and 6). Insulin and insulin-
like signaling have been shown to function independently of
the PI3K pathway in multiple systems, usually regulating the
ERK or the JNK pathway; thus, engagement ofthe ERK signaling
cascade by DAF-2 is not by itself surprising. In fact, studies in
humans indicate that circulating insulin triggers ovarian follicle
maturation by activating the IGFR1 in granulosa-luteal cells
and inducing progesterone secretion (reviewed in Silva et al.,
2009). Here, IGFR also appears to act independently of the
PI3K and AKT pathway, with speculation that it might instead
act either through the ERK or the JNK pathway (Poretsky et al.,
What was surprising, however, was that DAF-2 signaling was
still regulated by PTEN, which typically inhibits insulin signaling
by opposing the activity of PI3K (AGE-1) and converting PIP3
back into PIP2 via its phosphatase activity (Das et al., 2003). A
recent study also found that PTEN acts independently of PI3K
in a distinct region of the C. elegans germline. Brisbin et al.,
2009 showed that DAF-18/PTEN acts downstream of the
VAB-1 Eph RTK/MSP sperm signal to negatively regulate ovula-
tion independently of both PI3K and FOXO. Here, DAF-18 also
acts to negatively regulate RAS-MPK-1 signaling; in this
instance, via the sperm receptor. However, how might DAF-18
work in an AGE-1 independent pathway to regulate RAS sig-
naling downstream to DAF-2 in zone 1 of the germline? One pos-
sibility is that an as-yet-unidentified PI3K exists in the C. elegans
signal to the RAS-MPK-1 pathway; a second is that DAF-18
function may itself bemodified byphosphorylation as suggested
Figure 8. Nutritional Cues and DAF-2/LET-60 RAS/MPK-1 ERK SignalingPathway Regulate Zone 1MPK-1 Activation and Oocyte Production
in C. elegans Germline
(A) Nutritional cuessignal via the DAF-2 insulin-like receptor and result in activation of the RAS-MPK-1 in zone 1. MPK-1 activation inzone 1 drives progression of
meiotic prophase and oocyte production. The sperm signal activates MPK-1 in zone 2, which ensures oocyte maturation and ovulation. DTC, distal tip cell.
(B) In the absence of nutrition, the germline turns off the MPK-1 signal in zone 1, stalling meiotic prophase progression and oocyte production.
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
238 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
Angelo, G., and Van Gilst, M.R. (2009). Starvation protects germline stem cells
and extends reproductive longevity in C. elegans. Science 326, 954–958.
Antebi, A., Yeh, W.H., Tait, D., Hedgecock, E.M., and Riddle, D.L. (2000).
daf-12 encodes a nuclear receptor that regulates the dauer diapause and
developmental age in C. elegans. Genes Dev. 14, 1512–1527.
Apfeld, J., and Kenyon, C. (1998). Cell nonautonomy of C. elegans daf-2 func-
tion in the regulation of diapause and life span. Cell 95, 199–210.
Apfeld,J.,and Kenyon, C.(1999).Regulationoflifespanby sensory perception
in Caenorhabditis elegans. Nature 402, 804–809.
Arur, S., Ohmachi, M., Nayak, S., Hayes, M., Miranda, A., Hay, A., Golden, A.,
and Schedl, T. (2009). Multiple ERK substrates execute single biological
processes in Caenorhabditis elegans germ-line development. Proc. Natl.
Acad. Sci. USA 106, 4776–4781.
Bargmann, C.I., and Horvitz, H.R. (1991). Control of larval development by
chemosensory neurons in Caenorhabditis elegans. Science 251, 1243–1246.
Brisbin, S., Liu, J., Boudreau, J., Peng, J., Evangelista, M., and Chin-Sang, I.
(2009). A role for C. elegans Eph BTK signaling in PTEN regulation.
Developmental Cell 17, 459–469.
Brunet, S., and Maro, B. (2005). Cytoskeleton and cell cycle control during
meiotic maturation of the mouse oocyte: integrating time and space.
Reproduction 130, 801–811.
Cavaliere, V., Donati, A., Hsouna, A., Hsu, T., and Gargiulo, G. (2005). dAkt
kinase controls follicle cell size during Drosophila oogenesis. Dev. Dyn. 232,
Charlesworth, A., Ridge, J.A., King, L.A., MacNicol, M.C., and MacNicol, A.M.
(2002). A novel regulatory element determines the timing of Mos mRNA trans-
lation during Xenopus oocyte maturation. EMBO J. 21, 2798–2806.
Choi, T., Fukasawa, K., Zhou, R., Tessarollo, L., Borror, K., Resau, J., and
Vande Woude, G.F. (1996). The Mos/mitogen-activated protein kinase
(MAPK) pathway regulates the size and degradation of the first polar body in
maturing mouse oocytes. Proc. Natl. Acad. Sci. USA 93, 7032–7035.
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
Developmental Cell 27, 227–240, October 28, 2013 ª2013 Elsevier Inc. 239
Church, D.L., Guan, K.L., and Lambie, E.J. (1995). Three genes of the MAP
kinase cascade, mek-2, mpk-1/sur-1 and let-60 ras, are required for meiotic
cell cycle progression in Caenorhabditis elegans. Development 121, 2525–
Colombani, J., Raisin, S., Pantalacci, S., Radimerski, T., Montagne, J., and
Le ´opold, P. (2003). A nutrient sensor mechanism controls Drosophila growth.
Cell 114, 739–749.
Darwin, C. (1859). On the Origin of Species by Means of Natural Selection, or
the Preservation of Favoured Races in the Struggle for Life, Volume 1, First
Edition. (London: W. Clowes and Sons).
Das, S., Dixon, J.E., and Cho, W. (2003). Membrane-binding and activation
mechanism of PTEN. Proc. Natl. Acad. Sci. USA 100, 7491–7496.
Dorman, J.B., Albinder, B., Shroyer, T., and Kenyon, C. (1995). The age-1 and
daf-2 genes function in a common pathway to control the lifespan of
Caenorhabditis elegans. Genetics 141, 1399–1406.
eny respond to nutritional changes during Drosophila oogenesis. Dev. Biol.
Evans, E.A., Chen, W.C., and Tan, M.W. (2008). The DAF-2 insulin-like
signaling pathway independently regulates aging and immunity in C. elegans.
Aging Cell 7, 879–893.
Fouladi-Nashta, A.A., Gutierrez, C.G., Gong, J.G., Garnsworthy, P.C., and
Webb, R. (2007). Impact of dietary fatty acids on oocyte quality and develop-
ment in lactating dairy cows. Biol. Reprod. 77, 9–17.
Frank-Vaillant, M., Jessus, C., Ozon, R., Maller, J.L., and Haccard, O. (1999).
Two distinct mechanisms control the accumulation of cyclin B1 and Mos in
Xenopus oocytes in response to progesterone. Mol. Biol. Cell 10, 3279–3288.
Gumienny, T.L., Lambie, E., Hartwieg, E., Horvitz, H.R., and Hengartner, M.O.
(1999). Genetic control of programmed cell death in the Caenorhabditis
elegans hermaphrodite germline. Development 126, 1011–1022.
Hietakangas, V., and Cohen, S.M. (2009). Regulation of tissue growth through
nutrient sensing. Annu. Rev. Genet. 43, 389–410.
Hsu, H.J., and Drummond-Barbosa, D. (2009). Insulin levels control female
germline stem cell maintenance via the niche in Drosophila. Proc. Natl.
Acad. Sci. USA 106, 1117–1121.
Ivanovska, I., Lee, E., Kwan, K.M., Fenger, D.D., and Orr-Weaver, T.L. (2004).
Kenyon, C., Chang, J., Gensch, E., Rudner, A., and Tabtiang, R. (1993). A C.
elegans mutant that lives twice as long as wild type. Nature 366, 461–464.
LaFever, L., and Drummond-Barbosa, D. (2005). Direct control of germline
stem cell division and cyst growth by neural insulin in Drosophila. Science
Lakowski, B., and Hekimi, S. (1998). The genetics of caloric restriction in
Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 95, 13091–13096.
Lee, M.H., Ohmachi, M., Arur, S., Nayak, S., Francis, R., Church, D., Lambie,
E., and Schedl, T. (2007). Multiple functions and dynamic activation of MPK-1
extracellular signal-regulated kinase signaling in Caenorhabditis elegans
germline development. Genetics 177, 2039–2062.
Lin, K., Hsin, H., Libina, N., and Kenyon, C. (2001). Regulation of the
Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germ-
line signaling. Nat. Genet. 28, 139–145.
Matten, W.T., Copeland, T.D., Ahn, N.G., and Vande Woude, G.F. (1996).
Positive feedback between MAP kinase and Mos during Xenopus oocyte
maturation. Dev. Biol. 179, 485–492.
McCarter, J., Bartlett, B., Dang, T., and Schedl, T. (1997). Soma-germ cell
interactions in Caenorhabditis elegans: multiple events of hermaphrodite
germline development require the somatic sheath and spermathecal lineages.
Dev. Biol. 181, 121–143.
Michaelson, D., Korta, D.Z., Capua, Y., and Hubbard, E.J. (2010). Insulin
signaling promotes germline proliferation in C. elegans. Development 137,
Miller, M.A., Nguyen, V.Q., Lee, M.H., Kosinski, M., Schedl, T., Caprioli, R.M.,
and Greenstein, D. (2001). A sperm cytoskeletal protein that signals oocyte
meiotic maturation and ovulation. Science 291, 2144–2147.
Miller, M.A., Ruest, P.J., Kosinski, M., Hanks, S.K., and Greenstein, D. (2003).
An Eph receptor sperm-sensing control mechanism for oocyte meiotic matu-
ration in Caenorhabditis elegans. Genes Dev. 17, 187–200.
Neufeld, T.P. (2003). Shrinkage control: regulation of insulin-mediated growth
by FOXO transcription factors. J. Biol. 2, 18.
Ogg, S., and Ruvkun, G. (1998). The C. elegans PTEN homolog, DAF-18, acts
in the insulin receptor-like metabolic signaling pathway. Mol. Cell 2, 887–893.
Okumura, K., Zhao, M., DePinho, R.A., Furnari, F.B., and Cavenee, W.K.
(2005). PTEN: a novel anti-oncogenic function independent of phosphatase
activity. Cell Cycle 4, 540–542.
Papadopoulos, S., Lonergan, P., Gath, V., Quinn, K.M., Evans, A.C.,
O’Callaghan, D., and Bolan, M.P. (2001). Effect of diet quantity and urea
supplementation on oocyte and embryo quality in sheep. Theriogenology 55,
Pinkston, J.M.,Garigan, D., Hansen, M.,and Kenyon, C.(2006).Mutationsthat
increase the life span of C. elegans inhibit tumor growth. Science 313,
Plowman, G.D., Sudarsanam, S., Bingham, J., Whyte, D., and Hunter, T.
(1999). The protein kinases of Caenorhabditis elegans: a model for signal
transduction in multicellular organisms. Proc. Natl. Acad. Sci. USA 96,
Poretsky, L.,Cataldo, N.A., Rosenwaks,Z.,and Giudice,L.C. (1999). Theinsu-
lin-related ovarian regulatory system in health and disease. Endocr. Rev. 20,
Poretsky, L., Seto-Young, D., Shrestha, A., Dhillon, S., Mirjany, M., Liu, H.C.,
Yih, M.C., and Rosenwaks, Z. (2001). Phosphatidyl-inositol-3 kinase-indepen-
dent insulin action pathway(s) in the human ovary. J. Clin. Endocrinol. Metab.
Roy, L.M., Haccard, O., Izumi, T., Lattes, B.G., Lewellyn, A.L., and Maller, J.L.
(1996). Mos proto-oncogene function during oocyte maturation in Xenopus.
Oncogene 12, 2203–2211.
Sharma, K.K., Wang, Z., Motola, D.L., Cummins, C.L., Mangelsdorf, D.J., and
Auchus, R.J. (2009). Synthesis and activity of dafachronic acid ligands for the
C. elegans DAF-12 nuclear hormone receptor. Mol. Endocrinol. 23, 640–648.
Shen, Y., Wollam, J., Magner, D., Karalay, O., and Antebi, A. (2012). A steroid
receptor-microRNA switch regulates life span in response to signals from the
gonad. Science 338, 1472–1476.
Silva, J.R., Figueiredo, J.R., and van den Hurk, R. (2009). Involvement of
growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian
folliculogenesis. Theriogenology 71, 1193–1208.
Song, M.S., Carracedo, A., Salmena, L., Song, S.J., Egia, A., Malumbres, M.,
and Pandolfi, P.P. (2011). Nuclear PTEN regulates the APC-CDH1 tumor-
suppressive complex in a phosphatase-independent manner. Cell 144,
Sundaram, M.V. (2006). RTK/Ras/MAPK signaling. WormBook, 1–19.
Verlhac, M.H., de Pennart, H., Maro, B., Cobb, M.H., and Clarke, H.J. (1993).
MAP kinase becomes stably activated at metaphase and is associated with
microtubule-organizing centers during meiotic maturation of mouse oocytes.
Dev. Biol. 158, 330–340.
Verlhac, M.H., Kubiak, J.Z., Weber, M., Ge ´raud, G., Colledge, W.H., Evans,
M.J., and Maro, B. (1996). Mos is required for MAP kinase activation and is
involved in microtubule organization during meiotic maturation in the mouse.
Development 122, 815–822.
DAF-2 Couples Nutrition to ERK-Mediated Oogenesis
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