Developmental timing mechanisms are integrated with various
signaling pathways to achieve the synchrony and succession of
stage-specific programs during animal development. In C. elegans,
a network of heterochronic genes has been identified that control
developmental timing of diverse post-embryonic cell lineages, best
characterized by the development of a row of lateral hypodermal
seam cells, which undergo stage-specific developmental programs
during each of the four larval stages and terminally differentiate at
the late L4 larval stage (Ambros, 2000; Rougvie, 2005; Moss,
2007). Mutations in heterochronic genes cause skipping or
reiteration of stage-specific programs, resulting in premature or
delayed terminal differentiation of seam cells. Adult animals, the
somatic cells of which are post-mitotic, undergo a progressive
decline in pharyngeal pumping and body movement, and also
experience cell and tissue deterioration. Several mechanisms,
including caloric restriction, the insulin/insulin-like growth factor
1 (IGF-1) endocrine system and the steroid hormone system,
regulate aging in C. elegans (Guarente and Kenyon, 2000; Kenyon,
2005; Antebi, 2007). For example, reduced activity of daf-2, which
encodes an insulin/IGF-1 receptor, extends the lifespan (Kenyon,
2005). The steroid hormone pathway, acting via the nuclear
receptor DAF-12, also impacts the succession of developmental
events in larvae (Fielenbach and Antebi, 2008). Two components
of the heterochronic circuit, miRNA lin-4 and its target lin-14
(which encodes a nuclear transcription factor), also have a modest
effect on adult lifespan (Boehm and Slack, 2005). lin-4/lin-14 may
influence lifespan by regulating metabolic outputs, and the insulin-
like gene ins-33 is a direct target of LIN-14 (Hristova et al., 2005).
Nevertheless, it remains largely unknown whether genes
controlling larval developmental timing also function in the adult
and whether signals that modulate aging, such as insulin/IGF-1,
also act within the heterochronic circuit.
MATERIALS AND METHODS
The following strains were used in this study: LGI, lin-28(n719), daf-
16(mu86); LGII, lin-42(n1089), sea-1(gk799), sea-2(bp283), sea-
2(tm4355), lin-4(e912), lin-29(n333); LGIII, daf-2(e1370); LGIV, lin-
66(ku423), jcIs1(ajm-1::gfp), zIs356(daf-16::gfp); LGV, lin-46(bp312), lin-
46(bp284), wIs51(scm::gfp); and LGX, daf-12(rh257), daf-12(rh61rh411),
lin-14(n179), alg-1(gk214), ain-1(bp299). The genetic location for
muIs84(sod-3::gfp), bpIs124(dcap-1::rfp) and bpIs145(lin-28::gfp::lin-28
3?UTR(LCE)) was not determined.
lin-46(bp312) contains a glycine to stop codon mutation at amino acid
249. lin-46(bp284) was isolated in a screen for mutants that enhanced the
retarded heterochronic phenotype of sea-2(bp283). lin-46(bp284) contains
a leucine to phenylalanine mutation at amino acid 44 of LIN-46.
Isolation, characterization and cloning of bp283
Animals carrying the scm::gfp reporter were mutagenized and F2 progeny
were examined for the number of SCM::GFP-positive cells. From ~4000
genomes screened, 10 mutations were isolated that caused altered number
of seam cells. The sea-2 mutants are cold sensitive. At 15°C, the retarded
heterochronic defects are more severe. ccDf2/sea-2(bp283) mutants had an
average of 22.5 seam cells (n8) at 15°C, similar to 22.7 in sea-2(bp283)
mutants. All experiments were performed at 20°C unless otherwise noted.
bp283 was mapped by three factor mapping. From the sqt-2 lin-31 +/+
+ bp283 cross, 0 out of 32 Lin no Sqt recombinants carried sea-2(bp283).
From the lin-31 clr-1 dpy-10 +/+ + + bp283 cross, 0 out of 14 Lin non Clr
recombinants carried bp283. From the dpy-10 vab-19 +/+ + bp283 cross,
17 out of 39 Dpy non Vab recombinants carried bp283. Single nucleotide
polymorphism (SNP) mapping further located bp283 between pkp2115
(–6.31) and pkp2051 (–4.92). Fosmids covering this region were used for
transformation rescue experiments.
Development 138, 2059-2068 (2011) doi:10.1242/dev.057109
© 2011. Published by The Company of Biologists Ltd
National Institute of Biological Sciences, Beijing, 102206 Beijing, People’s Republic of
*Author for correspondence (email@example.com)
Accepted 14 February 2011
Like other biological processes, aging is regulated by genetic pathways. However, it remains largely unknown whether aging is
determined by an innate programmed timing mechanism and, if so, how this timer is linked to the mechanisms that control
developmental timing. Here, we demonstrate that sea-2, which encodes a zinc-finger protein, controls developmental timing in
C. elegans larvae by regulating expression of the heterochronic gene lin-28 at the post-transcriptional level. lin-28 is also essential
for the autosomal signal element (ASE) function of sea-2 in X:A signal assessment. We also show that sea-2 modulates aging in
adulthood. Loss of function of sea-2 slows the aging process and extends the adult lifespan in a DAF-16/FOXO-dependent
manner. Mutation of sea-2 promotes nuclear translocation of DAF-16 and subsequent activation of daf-16 targets. We further
demonstrate that insulin/IGF-1 signaling functions in the larval heterochronic circuit. Loss of function of the insulin/IGF-1 receptor
gene daf-2, which extends lifespan, also greatly enhances the retarded heterochronic defects in sea-2 mutants. Regulation of
developmental timing by daf-2 requires daf-16 activity. Our study provides evidence for intricate interplay between the
heterochronic circuit that controls developmental timing in larvae and the timing mechanism that modulates aging in adults.
KEY WORDS: sea-2, lin-28, Heterochronic genes, daf-2, daf-16, Aging, C. elegans
The zinc-finger protein SEA-2 regulates larval developmental
timing and adult lifespan in C. elegans
Xinxin Huang, Hui Zhang and Hong Zhang*
Development ePress online publication date 6 April 2011
Animals were placed on a 2% agarose pad in 5 l of M9 and the seam cell
division pattern was analyzed by microscopy. After observation, each
animal was rescued and placed on a plate to recover. This procedure was
repeated every 2 to 4 hours.
The PCR templates used for synthesizing RNA were: sea-2 (K10G6, nt
9550-10438); fox-1 (T07D1, nt 29314-30084); and sex-1 (F44A6, nt 11213-
12115). For RNA feeding of hbl-1, synchronized L1 worms were placed on
RNAi plates and worms from the next generation were examined.
Reporters for sea-2 were constructed by a PCR fusion based approach. The
fused PCR products were derived from two overlapping PCR fragments.
One contained the promoter region and the entire ORF of sea-2 (K10G6,
nt3055-16856), the other contained gfp and the unc-54 3?UTR or the sea-
2 3?UTR. The PCR products were co-injected with pRF4 [rol-6(su1006)]
into wild type animals and at least two stable transgenic lines were
analyzed. The sea-2::gfp::unc-54 3?UTR reporter displayed the same
expression pattern as the reporter containing the sea-2 3?UTR. We inserted
a nuclear localization signal sequence (PKKKRK) at the N terminus of
SEA-2 to determine whether SEA-2 acts in the nucleus or in the cytoplasm
to specify the temporal fates of seam cells. However, the sea-2::NLS::sea-
2::gfp transgene was expressed at a much lower level than sea-2::gfp,
indicating the addition of the NLS destabilized SEA-2.
lin-28::gfp::lin-28 3?UTR(LCE) contains the promoter and the entire
ORF of lin-28 (F02E9, nt 3769-7599), gfp and the lin-28 3?UTR (F02E9,
nt 3235-3765) with a deletion of the LCE (F02E9, nt 3424-3438). Animals
carrying the lin-28::gfp::lin-28 3?UTR(LCE) extrachromosomal array
were -ray irradiated and the resulting stable integrated line (bpIs145) was
outcrossed two times.
Lifespan and heat stress assay
The lifespan assay was performed at 20°C. Animals that had just passed the
final larval molt were transferred to new plates every 1-2 days until the end
of reproduction and 2-4 days thereafter. Animals were scored as dead when
they failed to response to gentle prodding. Worms with exploded vulva, or
that had bagged (died from internal hatching) or crawled off the plate were
excluded. Three independent assays were tested for each experiment.
GraphPad Prism 5 was used for survival curves and statistical analysis.
The heat stress assay was performed at 32°C using 1-day-old adults.
Animals were cultured and scored as described for lifespan assays. At least
100 animals were tested for each strain.
RNA isolation and real-time RT-PCR
Synchronized L1 and L3 animals were collected and total RNA was
extracted from about 500 animals using Trizol reagent (Sigma) according
to the manufacturer’s protocol. Total RNA (2 g) was reverse transcribed
using an Invitrogen Superscript III kit. Quantitative PCR reactions were
carried out using a SYBR RT-PCR kit (TaKaRa) and a Mastercycler ep
realplex machine (Eppendorf). eft-2 was used as an internal control (Bagga
et al., 2005). The level of lin-28 mRNA was normalized to the level of wild
type L1 worms, which was set to 1. Error bars indicate the standard
deviation (s.d.) of three independent experiments.
Primers used were: lin-28 FW, TCGGAGTCTTGATGAAGGAG; lin-
28 RW, GAGACAGCCTTCTTACGACC; eft-2 FW, ATGGTCAA -
CTTCACGGTCGATG; eft-2 RW: GATGGTAATACAACGCTCCTGC.
Fluorescence photography and quantification
Gut autofluorescence was photographed using a Zeiss Axioplan 2 imaging
system and quantified by AxioVision Rel. 4.6. The same exposure time
was used for different strains and also for animals of different ages.
Lysates from synchronized animals were prepared as previously described
(Seggerson et al., 2002), and endogenous LIN-28 was detected with diluted
anti-LIN-28 serum (1:2000) and HRP-conjugated goat anti-rabbit
secondary antibody. Anti-actin monoclonal antibody (A3853, Sigma) was
used as a gel loading control.
To determine the level of lin-4 RNA, total RNA isolation and northern
analyses were performed as described (Grishok et al., 2001). Total RNA
was isolated from synchronized L2 and L3 worms.
The following sequences were used to drive the expression of genomic
coding region of sea-2 and unc-54 3?UTR in various tissues: Pceh-16
(C13G5, nt 4341-6883), Pvha-6 (VW02B12L, nt 1023-2624), Pmyo-3
(WRM061aH08, nt 24240-26523) and Prgef-1(F25B3, nt 10721-14265).
At least three independent transgenic lines for each construct were
Mutations in sea-2 cause retarded heterochronic
Ten seam cells, aligned on each side of the animal, undergo
asymmetric cell division at each of four larval stages (L1 to L4)
with only one daughter retaining the seam cell identity. Certain
seam cells also undergo one round of symmetric division with
both daughter cells maintaining the seam cell fate at the L2
larval stage, increasing seam cell number from 10 at hatching
stage to 16 from L2 stage onwards. All seam cells terminally
differentiate at the late L4 stage, including cessation of cell
division, fusion with neighboring seam cells and synthesis of
adult-specific cuticular structures, called alae (Fig. 1A-C,G). In
a genetic screen to identify mutants with defective seam cell
development, we isolated a mutation, bp283, that increased the
number of seam cells in young adults from 16 in wild-type
animals to 20 (Table 1, Fig. 1D). Adult-specific alae were not
completely formed in bp283 mutant young adults, indicating a
delay in terminal differentiation of seam cells (Fig. 1E,F).
Analysis of seam cell lineages revealed that the L2 stage-specific
proliferative division pattern was reiterated at the L3 larval stage
in bp283 mutants and certain seam cells failed to fuse and
continued to divide at the L4/adult switch, resulting in an
increase in the seam cell number and discontinuous alae (Fig.
1H). The seam cell developmental defects in bp283 animals, as
in other classic heterochronic gene mutants, were completely
suppressed when animals developed through the alternate dauer
larval stage (Table 1). Thus, bp283 causes retarded heterochronic
Using transformation rescue we found that a transgene
containing a single gene, previously named sea-2 (see below),
rescued the heterochronic defects in bp283 mutants (Fig. 1I). We
sequenced cDNAs and found that sea-2 encodes a 1727 amino acid
protein. bp283 contains an alanine to aspartate mutation at codon
1267 (see Fig. S1 in the supplementary material). sea-2(tm4355),
which deletes amino acids 294 to 590 of SEA-2, showed retarded
heterochronic defects in sensitized genetic backgrounds (Table 1).
Animals bearing sea-2(bp283) in trans to ccDf2, a deficiency that
removes the sea-2 locus, had an average of 20.2 seam cells (n10),
compared with 20.4 in sea-2(bp283) mutants. sea-2(RNAi) also
caused retarded heterochronic defects, but did not further elevate
the defects in sea-2(bp283) mutants (Table 1). These results
indicate that sea-2(bp283) is probably a strong loss-of-function
Bioinformatic analysis revealed that SEA-2 contains four CCHC
zinc fingers and one CCHH zinc finger (see Fig. S1 in the
supplementary material). The zinc fingers Z1 and Z5 of SEA-2
strongly bound to single stranded (ss) and double stranded (ds)
RNA in an EMSA assay (see Fig. S2 in the supplementary
material). Transgenes expressing truncated SEA-2 with a deletion
Development 138 (10)
of the first zinc finger (amino acid 319-339) or the fifth zinc finger
(amino acid 1429-1449) failed to rescue the retarded heterochronic
defects in sea-2 mutants (see Table S1 and Fig. S3 in the
supplementary material), suggesting that the RNA binding domains
are important for SEA-2 function.
sea-2 is widely expressed
We constructed a reporter with gfp inserted at the C terminus of the
sea-2-coding region to determine the expression pattern of sea-2.
This translational reporter rescued the heterochronic defect in sea-
2 mutants (data not shown). sea-2 was strongly expressed in
various tissues, including seam cells, intestine cells, pharyngeal
muscles and nerve ring neurons (Fig. 1J-N). SEA-2::GFP
expression persisted into adulthood. SEA-2::GFP was diffusely
localized in both cytoplasm and nucleus (Fig. 1J-M).
sea-2 functions cell-autonomously to specify
temporal fates of seam cells
To determine whether sea-2 acts cell-autonomously in
controlling the stage specific fates of seam cells, we expressed
sea-2 using a seam cell-specific ceh-16 promoter (Huang et al.,
2009). sea-2 mutants carrying a ceh-16::sea-2 transgene had an
average of 16.3 seam cells (see Table S1 in the supplementary
material). Expression of sea-2 in the intestine, body wall muscle
cells or neurons failed to rescue the increased number of seam
cells in sea-2 mutants (see Table S1 and Fig. S3 in the
Mutations in sea-2 enhance other retarded
heterochronic mutations that cause reiteration of
the L2 stage-specific fate
To further characterize the role of sea-2 in specifying the L2/L3
progression, we examined the genetic interaction between sea-2
and other heterochronic mutants. Other mutants that cause
reiteration of the L2 stage fate at the L3 stage and an incomplete
defect in the larval/adult switch phenotype include the recessive
gain of function (rh257) or null allele (rh61 rh411) of daf-12 and
loss of function of lin-46 (the gephyrin homolog), lin-66 (a novel
protein), alg-1 (the Argonaute homolog), ain-1 (the GW182
homolog) and let-7 family miRNAs (Moss, 2007). sea-2(bp283)
in combination with a mutation in each of these retarded
heterochronic genes caused a dramatic increase in seam cell
Role of sea-2 in developmental timing and lifespan
Fig. 1. Mutations in sea-2 cause retarded
heterochronic defects. (A-C)In a wild-type
young adult animal, 16 seam cells are present
on each side (A). Longitudinal cuticular ridges,
known as alae, are synthesized by seam cells
and run continuously from head to tail (arrow,
B). Seam cells fuse together (arrow, C), marked
by the adherens junction marker ajm-1::gfp.
The number of seam cells is indicated in
parentheses. Scale bar: 20m. (D-F)In sea-2
mutant young adult animals, the number of
seam cells is increased (D) and certain seam
cells fail to terminally differentiate, resulting in
gaps in the alae (arrow, E) and defective fusion
with neighboring seam cells (arrow, F). Scale
bars: 20m. (G)The seam cell lineage from the
L1 to young adult stage in a wild-type animal.
Squares represent the fusing daughter cells
and the three horizontal lines at the bottom of
the lineage stand for adult alae formation.
(H)Seam cell lineage of a sea-2 mutant grown
at 15°C. Certain seam cells repeat the
proliferative division pattern at the L3 stage
(highlighted in red). Twelve sea-2 animals were
analyzed and the number of seam cells that
repeated the L2 division pattern at the L3
stage varied among individual sea-2 animals,
which was consistent with the range of seam
cell numbers present in sea-2 mutants.
(I)Cloning of sea-2. sea-2 was mapped on
chromosome II (LGII), close to lin-31. Fosmid
WRM0635dA01 (in all seven transgenic lines
examined) and the DNA fragment covering
K10G6.3 (in all four transgenic lines examined)
rescued the retarded heterochronic defects in
sea-2 mutants. The genomic structure of sea-2
is shown at the bottom. (J)Expression of sea-
2::gfp in an L3 larva. Scale bar: 20m.
(K-N)Expression of sea-2 in the head region
(K), tail region (L) and seam cells (arrows in M).
(N)Nomarski image of the animal shown in M.
Scale bars: 20m.
numbers and a much more complete terminal differentiation
defect (Table 1, Fig. 2A-D). For example, sea-2; lin-66 double
mutants had an average of 90 seam cells at the young adult stage,
compared with 20 in sea-2 and 37 in lin-66 single mutants. In
sea-2; lin-66 mutants the L2 stage program was reiterated at
both the L3 and L4 stages (Fig. 2E). The mutant alleles used are
strong loss of function or null. The genetic interactions suggest
that sea-2 probably functions in parallel to these genes in
specifying the L2/L3 switch.
We further examined the relationship between sea-2 and other
retarded heterochronic mutants. The lin-4(e912) null mutation leads
to the reiteration of the L1 stage fate at subsequent larval stages. lin-
29 functions downstream in the heterochronic pathway in specifying
the larval/adult switch (Ambros and Horvitz, 1984). We found that
as in lin-4 and lin-29 single mutants, no alae were generated at the
young adult stage in sea-2 lin-4 and sea-2 lin-29 double mutants (see
Table S2 in the supplementary material). This is consistent with the
hypothesis that sea-2 functions upstream of lin-29.
The heterochronic defect in sea-2 mutants is
suppressed by loss of function of lin-28
To place sea-2 in the heterochronic pathway, we examined the
phenotype of sea-2 mutants combined with precocious
heterochronic mutants. lin-28, which encodes a protein with a cold
shock domain and a CCHC zinc finger, specifies the L2 program
(Moss et al., 1997). In lin-28 mutants, L2-stage events are skipped
and the larval/adult switch takes place at the L3 stage (Fig. 3A,B)
(Ambros and Horvitz, 1984). The retarded heterochronic defects in
sea-2 mutants were completely suppressed by lin-28. lin-28; sea-
2 double mutants showed the same seam cell development
phenotype as lin-28 single mutants (Table 1; Fig. 3C,D). hbl-1 (the
Hunchback homolog) regulates the L2 fate and also the L4/adult
switch. Loss of function of hbl-1 causes a precocious heterochronic
phenotype and also suppresses the retarded heterochronic defect in
let-7 family miRNAs mutants (Abrahante et al., 2003; Lin et al.,
2003; Abbott et al., 2005). We found that sea-2(bp283) partially
suppressed the precocious heterochronic defect in hbl-1(RNAi)
Development 138 (10)
Table 1. Role of sea-2 and daf-2 signaling in the heterochronic pathway
Formation of alae (%)
at the young adult stage
Formation of alae
(%) at the L3 stage (n)
Number of seam cells
in young adults (n) Genotype NoPartial Full
sea-2(bp283) from dauer
mir-48 mir-241(nDf51); mir-84(n4037)
sea-2(RNAi); mir-48 mir-241(nDf51); mir-84(n4037)
daf-16(mu86); sea-2(bp283); daf-2(e1370)
*sea-2(RNAi) was delivered by injection.
†bpIs145: lin-28::gfp::lin-28 3’UTR(LCE).
no, no alae; partial, alae gaps; full, complete alae; ND, not determined.
n, number of animal sides examined.
hbl-1 plays a secondary, opposite role during adulthood. hbl-1(RNAi) causes one or more breaks in adult alae and also abnormal nuclear division of seam cells at the young
adult stage (Lin et al., 2003). Thus, the number of seam cells and the formation of alae in hbl-1(RNAi) animals were not examined.
animals (Table 1). The sea-2 mutation did not affect temporal
expression of hbl-1 (see Fig. S4 in the supplementary material).
sea-2 mutants also partially suppressed other precocious mutants,
including lin-14 and lin-42 (see Table S2 in the supplementary
material). These genetic analyses suggest that sea-2 functions
through lin-28 in specifying the L2/L3 progression.
lin-28 is ectopically expressed in sea-2 mutants
To understand how sea-2 regulates lin-28 activity, we examined
expression of the translational fusion reporter lin-28::gfp::lin-28
3?UTR, which contains the lin-28 coding and regulatory region. This
transgene rescues the mutant phenotype of lin-28(n719) animals
(Moss et al., 1997). lin-28::gfp is expressed in diverse cell types,
including cells in the head, tail, muscles and seam cells. In wild-type
animals, lin-28::gfp is expressed in L1 larvae, is detectable but
diminished in L2 larvae and is almost undetectable from the L3 stage
onwards (Fig. 4A,D; data not shown) (Moss et al., 1997). However,
we found that in sea-2 mutants high levels of LIN-28::GFP persisted
in the head and tail at the L3 and L4 larval stages (Fig. 4B-D). The
reporter also showed expression in seam cells in 87.5% of sea-2
mutant L3 larvae (n16) (Fig. 4E,F), whereas its expression was not
detected in seam cells in wild type L3 larvae (n15).
We further performed an immunoblot assay to examine the
levels of endogenous LIN-28 protein using an anti-LIN-28
antibody. In wild-type animals, LIN-28 protein was present at the
L1 larval stage, but was greatly reduced at the L3 larval stage (Fig.
4G) (Seggerson et al., 2002; Morita and Han, 2006). In sea-2
mutants, levels of LIN-28 remained high in L3 larvae (Fig. 4G).
Upregulation of LIN-28 in sea-2 mutants was more prominent than
that in daf-12 mutants (Fig. 4G). We conclude that mutations in
sea-2 cause ectopic expression of lin-28.
sea-2 regulates lin-28 expression at the
We next determined at what level sea-2 regulates lin-28 expression.
No upregulation of lin-28 mRNA was observed in L1 and L3 larvae
in sea-2 mutants (Fig. 4H); rather, levels of lin-28 transcripts were
even lower than wild type (Fig. 4H). This could be because wild-type
SEA-2 affects the transcription or stability of lin-28 or because high
levels of LIN-28 in sea-2 mutants negatively regulate the lin-28
mRNA levels. To explore whether sea-2 regulates lin-28 expression
at the post-transcriptional level via its 3?UTR, we examined
expression of the col-10::lacZ::lin-28 3?UTR reporter (pKM50), in
which the expression of lacZ is driven by the hypodermal cell-
specific promoter col-10 (Fig. 4I) (Morita and Han, 2006). In wild-
type animals, expression of this reporter was strong in L1 larvae but
absent in adults (Fig. 4I,J and data not shown). High expression
persisted, however, in sea-2 mutant adults (Fig. 4I,J). The col-
10::lacZ::unc-54 3?UTR reporter, in which the unc-54 3?UTR was
used instead of lin-28 3?UTR, was highly expressed in both wild
type and sea-2 mutants at the young adult stage (pKM53, Fig. 4J),
suggesting that sea-2 represses lin-28 expression through its 3?UTR.
To identify the SEA-2 response element, we examined the
expression of a series of reporters with deletions of discrete
regulatory elements in the lin-28 3?UTR. sea-2(bp283) dramatically
increased expression of a reporter with a mutation in the putative
Role of sea-2 in developmental timing and lifespan
Fig. 2. Loss of function of sea-2 causes strong synergistic
heterochronic defects in lin-66 mutants. (A-D)The number of seam
cells in an L2 (A), L3 (B), L4 (C) and young adult (D) sea-2; lin-66 double
mutant. The number of seam cells is indicated in parentheses. Scale
bars: 20m. (E)Schematic summary of the differentiation pattern of
certain seam cells in sea-2; lin-66 mutants. The L2 division pattern is
repeated at the L3 and L4 stages in sea-2; lin-66 mutants (highlighted
in red). At the young adult stage, seam cells continue to divide and fail
to form alae.
Fig. 3. Loss of function of lin-28 completely suppresses sea-2
heterochronic defects. (A,B)Loss of function of lin-28 causes
precocious heterochronic defects. In lin-28 mutants, the L2 division
pattern is skipped and thus fewer seam cells are present at the young
adult stage (A) and alae are precociously formed at the L3 molt (arrow,
B). Scale bars: 20m. (C,D)The retarded heterochronic phenotype in
sea-2 mutants is completely suppressed by loss of function of lin-28.
The seam cell number is reduced (C) and alae are precociously formed
at the L3 stage (arrow, D) in lin-28; sea-2 mutants. Scale bar: 20m.
DAF-12 response element (pKM63) (Fig. 4J) (Morita and Han,
2006). However, sea-2(bp283) did not increase the expression of a
col-10::lacZ::lin-28 transgene lacking the lin-4 binding site
(pKM55) (Fig. 4J). A reporter lacking the lin-4 complementary
element (LCE), lin-28::gfp::lin-28 3?UTR(LCE), is strongly
expressed at late larval stages and also causes retarded heterochronic
defects (see Fig. S5 in the supplementary material) (Moss et al.,
1997). sea-2(bp283) did not further elevate reporter expression or the
retarded heterochronic defects in animals carrying lin-28::gfp::lin-
28 3?UTR(LCE) (Table 1 and data not shown). Compared with
wild-type animals, levels of lin-4 remained unchanged at the L2 and
L3 larval stage in sea-2 mutants (see Fig. S5 in the supplementary
material). These results indicate that sea-2 probably acts through the
LCE to regulate lin-28 expression at the post-transcriptional level.
lin-28 is essential for the ASE function of sea-2 in
X:A signal assessment
sea-2 was previously identified as an autosomal signal element
(ASE), loss of function of which suppresses the XX lethality
phenotype caused by simultaneous depletion of the X signal
Development 138 (10)
Fig. 4. sea-2 regulates expression of lin-28 at the post-
transcriptional level through its 3? ?UTR. (A)In wild-type
animals, the lin-28::gfp reporter is not detectable at the L3
stage in the head region (A) or the tail region (not shown).
Scale bars: 20m. (B,C)High expression level of lin-28::gfp
persists at the L3 stage in the head region (B) and the tail
region (C) in sea-2 mutants (arrow). Irregular fluorescence
particles in C are gut autofluorescence. Scale bar: 20m.
(D)Percentage of wild type and sea-2 mutant animals
expressing the lin-28::gfp reporter at different larval stages.
Number of animals examined: wild type: L2 (n22), L3 (n34)
and L4 (n24); sea-2, L2 (n29), L3 (n34) and L4 (n26).
(E,F)Expression of the lin-28::gfp reporter in seam cells (arrow)
in sea-2 mutant L3 larvae. (F)DIC image of the seam cell
shown in E. (E,F)Confocal images. Scale bar: 10m.
(G)Western blot assay of endogenous LIN-28 protein using an
anti-LIN-28 antibody. Arrows indicate LIN-28 protein bands
due to alternative splicing as previously reported (Seggerson et
al., 2002; Morita and Han, 2006). lin-28(n719) was used as a
negative control. (H)Levels of lin-28 mRNA, detected by
quantitative RT-PCR, in wild type and sea-2 mutants at the L1
and L3 stage. Consistent with published data, lin-28
transcripts decrease from the L1 to the L3 stage in wild-type
animals (Morita and Han, 2006). Error bars indicate the s.d.
(I)Schematic structure of col-10::lacZ::lin-28 reporter (pKM50).
The DAF-12 response element (red box) and the lin-4
complementary site LCE (box) are indicated. pKM50 is hardly
expressed at the young adult stage in wild-type animals, but is
strongly expressed in sea-2 mutants. Scale bar: 100m.
(J)Percentage of animals showing the expression of reporters
in wild type and sea-2 mutants. pKM53: col-10::lacZ::unc-54
3?UTR. pKM55 lacks the lin-4 binding site in the lin-28 3?UTR.
pKM63 [col-10::lacZ::lin-28 3?UTR (caaa to accc)] harbors a 4
bp substitution (caaa to accc) in the lin-28 3?UTR. Number of
animals examined: wild type, pKM50 (n309), pKM53
(n398), pKM55 (n220) and pKM63 (n133); sea-2, pKM50
(n265), pKM53 (n432), pKM55 (n278) and pKM63
(n267). (K)lin-28 is required for the role of sea-2, but not
sea-1, in suppressing the XX lethality of fox-1 sex-1. Number
of embryos examined: wild type (n238), sea-2 (n232), lin-
28; sea-2 (n219), sea-1 (n220), lin-28; sea-1 (n217) and
elements (XSEs) sex-1 and fox-1 (Meyer, 2005) (P. Nix and B.
Meyer, personal communication). ASEs (also known as denominator
elements) function with XSEs (also known as numerator elements)
to communicate the ratio of X chromosomes to sets of autosomes
(X:A signal) to determine sexual fate and to equalize expression of
X-linked genes between hermaphrodites (XX) and males (XO)
(Meyer, 2005). Thus, we determined whether sea-2 also functions
through lin-28 to suppress the lethality of fox-1 sex-1 mutants in XX
animals. Simultaneously depleting the activity of lin-28 in sea-2; fox-
1 sex-1 hermaphrodites caused XX animals to arrest during
embryogenesis (Fig. 4K). lin-28 has no effect on the suppression of
the lethality of fox-1 sex-1 by loss of function of another ASE, sea-
1 (Powell et al., 2005) (Fig. 4K). Mutations in other heterochronic
genes, including lin-14, daf-12 and hbl-1 on the X chromosome, had
no effect on the ASE function of sea-2 (see Table S3 in the
supplementary material). Loss of function of fox-1 sex-1 still caused
embryonic lethality in animals carrying the lin-28::gfp::lin-28
3?UTR(LCE) transgene (Fig. 4K), indicating that lin-28 is
necessary but not sufficient to mediate the role of sea-2 in the sex
determination and dosage compensation pathways.
sea-2 mutants have an extended lifespan
We next examined whether sea-2 functions in adult animals.
Compared with wild-type animals, sea-2(bp283) mutants had a
significant increase in lifespan (Fig. 5A; see Table S4 in the
supplementary material). To determine whether sea-2 mutants had a
slower aging process, we examined two age-related markers, the
accumulation of lipofuscin fluorescence in intestine cells (Garigan et
al., 2002) and the accumulation of DCAP-1 (mRNA decapping
enzyme)-labeled cytoplasmic processing bodies (P bodies). P bodies
contain a variety of ribonucleoproteins and serve as sites for mRNA
turnover and storage (Parker and Sheth, 2007). We found that
DCAP-1 bodies gradually increased in hypodermal and muscle cells
in aged wild-type animals (see Fig. S6 in the supplementary
material). The accumulation of DCAP-1 bodies was slightly
accelerated in short-lived daf-16 mutants, and greatly decreased in
long-lived daf-2 mutants (Fig. 5B-D). sea-2 mutants accumulated
DCAP-1 bodies and lipofuscin fluorescence more slowly than wild-
type animals (Fig. 5E-K). Many long-lived C. elegans mutants are
resistant to heat stress (Kenyon, 2005). After heat shock treatment,
sea-2 mutants also survived longer than wild-type animals (Fig. 5L).
The decreased rate of age-dependent reporter accumulation and
elevated heat stress resistance confirm that loss of function of sea-2
slows the rate of aging and extends the adult lifespan.
The extended lifespan in sea-2 mutants depends
DAF-16, a FOXO family transcription factor, is a master regulator
of adult lifespan that integrates multiple inputs, including
insulin/IGF-1 signaling, increased dosage of sir-2.1 (the C. elegans
Role of sea-2 in developmental timing and lifespan
Fig. 5. sea-2 mutants have extended lifespan. (A)sea-2 mutants have extended lifespan. The long-lived phenotype of sea-2 is suppressed by loss of
function of daf-16. P0.0027 (<0.01) when comparing N2 and sea-2 mutants; P<0.001 when comparing daf-16; sea-2 and sea-2 mutants.
(B-E)Expression of DCAP-1::RFP in 4-day-old adults. Animals were photographed on the same day under identical conditions. Scale bar: 100m.
(F)Summary of relative signal intensity of DCAP-1::RFP. P0.285 when comparing daf-16 and wild type. P0.006 when comparing sea-2 and wild type.
P0.002 when comparing daf-2 and wild type. (G-J)The gut autofluorescence intensity in sea-2 mutants is weaker than in wild-type animals of the
same age. Photographs were taken at 100? magnification. Scale bars: 100m. (K)Summary of signal intensity of lipofuscin autofluorescence in wild
type and sea-2 mutants. P0.006 at day 8 when comparing sea-2 and wild type, while P0.003 at day 12. (L)sea-2 mutants are more resistant to heat
stress. The elevated stress resistance phenotype of sea-2 mutants is suppressed by daf-16. P<0.001 when comparing wild type and sea-2 mutants.
SIR2 NAD+-dependent protein deacetylase homolog) and reduced
activity of lin-14 (Boehm and Slack, 2005; Lin et al., 1997; Ogg et
al., 1997; Tissenbaum and Guarente, 2001). We thus investigated
whether sea-2 modulates lifespan through daf-16. daf-16; sea-2
double mutants had the same lifespan as daf-16 single mutants
(Fig. 5A; see Table S4 in the supplementary material). The heat
stress resistance of sea-2 mutants was also abolished by the daf-16
mutation (Fig. 5L). Thus, loss of activity of daf-16 suppressed the
longevity of sea-2 mutants. Compared with sea-2 and daf-2 single
mutants, the lifespan was further extended in sea-2; daf-2 double
mutants (Fig. 6A). The long-lived phenotype of sea-2; daf-2 double
mutants was also completely suppressed by loss of function of daf-
16 (Fig. 6A; see Table S4 in the supplementary material). Thus,
sea-2 probably acts in parallel to daf-2 signaling and converges on
daf-16 in regulating lifespan.
In wild-type young adult animals, DAF-16 is diffusely localized
in the cytoplasm (Fig. 6B). Reduced activity of insulin/IGF-l
signaling promotes nuclear translocation of DAF-16, which
subsequently activates expression of genes involved in modulating
stress resistance and aging (Lin et al., 1997; Ogg et al., 1997; Lee
et al., 2001; Lee et al., 2003). In sea-2 mutants, prominent nuclear
localization of DAF-16 was observed in cells of the intestine (Fig.
6C), a tissue that is important for mediating the effect of DAF-16
on aging (Libina et al., 2003). Weak nuclear localization of DAF-
16 was also evident in hypodermal and muscle cells (Fig. 6C). We
further examined the expression of sod-3, a well-characterized
target of DAF-16, in sea-2 mutants (Libina et al., 2003). In wild-
type young adult animals, sod-3::gfp was weakly expressed in
intestinal, hypodermal and pharyngeal cells (Fig. 6D,E).
Expression of sod-3::gfp was dramatically elevated in sea-2
mutants (Fig. 6F,G), but this upregulation was completely
abolished by reduced activity of daf-16 (Fig. 6H,I). Therefore, loss
of function of sea-2 results in nuclear translocation of DAF-16 and
activation of DAF-16 targets.
To investigate whether lifespan extension in sea-2 mutants
results from the retarded heterochronic defects at larval stages or is
determined at the adult stage, we measured the lifespan of animals
with reduced sea-2 activity at different ages. RNAi inactivation of
sea-2 at early larval stages caused retarded heterochronic defects
and also increased the lifespan (Table 1; data not shown). RNAi
inactivation of sea-2 in young adults had no effect on the temporal
fate of seam cells, but still extended the lifespan (Fig. 6J; see Table
S4 in the supplementary material). Therefore, sea-2 regulates
lifespan independent of its role in specifying developmental timing
Development 138 (10)
Fig. 6. sea-2 regulates adult lifespan in a DAF-16 dependent
manner. (A)Loss of function of sea-2 extends the lifespan of daf-
2(e1370) mutants. P0.002 when comparing sea-2; daf-2 and daf-2.
(B)Wild-type animals show cytoplasmic localization of DAF-16::GFP.
0% of wild-type animals (n73) show nuclear localization of DAF-
16::GFP. (C)Loss of function of sea-2 promotes nuclear localization of
DAF-16::GFP (arrows). 82.7% of sea-2 mutant animals (n75) show
nuclear localization of DAF-16::GFP. Scale bar: 20m. (D,E)Expression
of sod-3::gfp in wild-type adult animals. Weak GFP signal is detected in
the gut, pharynx and head neurons. (F,G)Loss of function of sea-2
elevates sod-3::gfp expression in hypodermal and gut cells in sea-2
adults. (H,I)Loss of function of daf-16 suppresses the enhanced
expression of sod-3::gfp in sea-2 mutants. (D,F,H) Same magnification.
Scale bars: 100m. (E,G,I) Same magnification. Scale bars: 20m.
(J)Reduction of sea-2 activity by RNAi feeding at the young adult stage
extends the lifespan. L4440: feeding with the empty vector. P<0.001.
(K)sea-2 acts in the intestine to regulate the lifespan. Expression of sea-
2 in the intestine, but not in seam cells, rescues the extended lifespan
phenotype in sea-2 mutants. (L)Model for the role of sea-2 in the
pathways that control developmental timing, aging and dose
sea-2 acts in the intestine to regulate lifespan
We next expressed sea-2 in a tissue-specific fashion to determine
whether sea-2 activity in any single tissue was sufficient to affect
the lifespan. sea-2 was specifically expressed in seam cells,
neurons, body wall muscles or the intestine by fusing it with the
ceh-16, rgef-1, myo-3 or vha-6 promoters, respectively (see Fig. S3
in the supplementary material). The lifespan of sea-2 mutants
carrying the transgene was measured. We found that expression of
sea-2 in the intestine, but not in other tissues, rescued the extended
lifespan phenotype in sea-2 mutants (Fig. 6K; see Table S5 in the
supplementary material). Therefore, sea-2 appears to act in the
intestine to regulate adult lifespan.
Loss of function of daf-2 causes heterochronic
defects in larvae
Finally, we investigated whether insulin/IGF-1 signaling plays a role
in the heterochronic circuit in larvae. daf-2 and daf-16 single mutants
had the wild-type number of 16 seam cells and displayed no evident
heterochronic defects (Table 1). However, daf-2(e1370) greatly
enhanced the retarded heterochronic defects in sea-2, daf-12 or alg-
1 mutants (Table 1). For example, the average number of seam cells
was increased from 20 in sea-2 mutants to 28 in sea-2; daf-2 mutants
and 42% of sea-2; daf-2 double mutants showed defective terminal
differentiation of seam cells, compared with 6% of sea-2 single
mutants. Loss of function of daf-16 did not suppress the
heterochronic defects in sea-2 mutants (Table 1). However, daf-16
completely suppressed the daf-2-dependent enhancement of the
heterochronic defects in sea-2 mutants (Table 1). Thus, daf-16 also
functions downstream of daf-2 in controlling developmental timing
in larvae. Expression of the lin-28::gfp reporter in sea-2 mutants was
not further elevated by simultaneous depletion of daf-2 (see Fig. S7
in the supplementary material). The hbl-1::gfp reporter was also
unchanged in sea-2; daf-2 mutants (see Fig. S7 in the supplementary
material). Thus, it remains to be determined whether daf-2 signaling
converges on lin-28 or hbl-1 in the heterochronic circuit.
Here, we demonstrated that the RNA-binding protein SEA-2
regulates the expression of lin-28 in the heterochronic circuit,
probably via the miRNA lin-4 complementary element in the
3?UTR. SEA-2 may modulate the function of the miRNA-induced
silencing complex (miRISC) at the lin-28 3?UTR. SEA-2 is not
generally involved in modulating miRNA activity, because loss of
function of sea-2 has no effect on expression of two other miRNA
targets, hbl-1 (regulated by let-7 family miRNAs) and cog-1
(regulated by lsy-6 miRNA) (see Fig. S4 in the supplementary
material and data not shown). The role of sea-2 in suppressing the
XX lethality of fox-1 sex-1 animals also requires the activity of lin-
28. Mammalian Lin28 functions as a post-transcriptional repressor
of the biogenesis of let-7 family miRNAs (Heo et al., 2008), raising
the possibility that LIN-28 mediates the biogenesis of miRNAs
(e.g. let-7) that in turn play a role in sex determination and dose
compensation. We also found that sea-2 acts in parallel to daf-2
insulin signaling to activate daf-16. Whether sea-2 regulates adult
lifespan through lin-28 remains uncertain. lin-28 mutants are short-
lived (data not shown), which could be due to pleiotropic defects
caused by loss of function of lin-28, including defective egg-laying.
Animals carrying the lin-28::gfp::lin-28 3?UTR(LCE) transgene
that showed elevated levels of lin-28 are also short-lived (data not
shown). Overexpression of sir-2.1 extends the adult lifespan in a
manner dependent on DAF-16. However, sea-2; sir-2.1(O/E)
animals are short-lived, which may be because double mutants
display multiple defects such as bursting vulva (data not shown).
Thus, the way in which sea-2 converges on daf-16 in regulating
lifespan has yet to be determined. Consistent with a study showing
that dauer arrest is decoupled from lifespan regulation, even though
both processes are controlled by IGF-1 signaling (Dillin et al.,
2002), sea-2 mutation alone has no effect on dauer formation nor
does it potentiate dauer formation in daf-2 mutants (data not
shown). Loss of function of daf-16 had no effect on the role of sea-
2 either in the heterochronic pathway or in suppressing fox-1 sex-
1 lethality (see Table S3 in the supplementary material). Therefore,
sea-2 acts through distinct effectors to regulate developmental
timing, dose compensation and lifespan (Fig. 6L). The multiple
functions of sea-2 during embryonic and larval development, such
as specifying the temporal fate of seam cells and functioning as an
ASE in dose compensation, indicate that wild-type sea-2 has
beneficial effects early in animal development. Thus, sea-2 is
favored by selection during evolution, even though sea-2 mutants
are long-lived. This provides support for the antagonistic pleiotropy
theory of aging (Hughes and Reynolds, 2005).
Our study further supports the presence of an intrinsic timing
mechanism that temporally initiates a program of aging in the adult,
consistent with a coordinate shift in gene expression pattern early in
adulthood in C. elegans and Drosophila (McCarroll et al., 2004).
Here, we have revealed a novel function of daf-2 insulin/IGF-1
signaling in controlling developmental timing at larval stages.
Reduced activity of daf-2 dramatically enhances retarded
heterochronic defects by causing reiteration of the L2 stage fate at
late larval stages. On the other hand, several components of the
heterochronic circuit also influence the rate of aging. Reducing lin-
14 activity or overexpressing lin-4 modestly extends lifespan in a
daf-16-dependent fashion (Boehm and Slack, 2005). However, there
is no correlation between the degree or type of heterochronic defect
and the rate of aging. lin-14 specifies the L1/L2 transition, while
sea-2 and daf-2 control the L2/L3 switch. Loss of function of lin-
14 causes precocious, whereas sea-2 and daf-12 mutations cause
retarded, heterochronic defects. lin-14 and sea-2 mutants have an
extended lifespan, whereas null daf-12 mutants have slightly
shortened lifespans (Fisher and Lithgow, 2006). sea-2, lin-14 and
daf-2 influence aging during adulthood in a way that is temporally
separable from their role in determining developmental timing
(Boehm and Slack, 2005; Dillin et al., 2002). Thus, the timing
program that modulates the aging process in adults shares a subset
of genes with the heterochronic circuit that functions at larval stages.
We thank Dr Barbara Meyer for comments on the manuscript, Drs Victor
Ambros and Eric Moss for the lin-28::gfp reporter and anti-LIN-28 antibody, Dr
David Fay for the hbl-1::gfp reporter, Dr Min Han for col-10::lacZ::lin-28
plasmids, Dr Shohei Mitani for sea-2(tm4355), and Dr Isabel Hanson for proof-
reading services. Some strains used in this work were received from the
Caenorhabditis Genetics Center. This work was supported by a Ministry of
Science and Technology grant (2010CB835201) to H.Z.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material for this article is available at
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Development 138 (10)