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Effect of life history on microRNA expression during C. elegans

development
Xantha Karp, Molly Hammell, Maria C. Ow, et al.

RNA published online February 22, 2011

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Effect of life history on microRNA expression during

C. elegans development

XANTHA KARP,1 MOLLY HAMMELL,2 MARIA C. OW, and VICTOR AMBROS

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA

ABSTRACT
Animals have evolved mechanisms to ensure the robustness of developmental outcomes to changing environments. MicroRNA
expression may contribute to developmental robustness because microRNAs are key post-transcriptional regulators of
developmental gene expression and can affect the expression of multiple target genes. Caenorhabditis elegans provides an
excellent model to study developmental responses to environmental conditions. In favorable environments, C. elegans larvae
develop rapidly and continuously through four larval stages. In contrast, in unfavorable conditions, larval development may be
interrupted at either of two diapause stages: The L1 diapause occurs when embryos hatch in the absence of food, and the dauer
diapause occurs after the second larval stage in response to environmental stimuli encountered during the first two larval stages.
Dauer larvae are stress resistant and long lived, permitting survival in harsh conditions. When environmental conditions
improve, dauer larvae re-enter development, and progress through two post-dauer larval stages to adulthood. Strikingly, all of
these life history options (whether continuous or interrupted) involve an identical pattern and sequence of cell division and cell
fates. To identify microRNAs with potential functions in buffering development in the context of C. elegans life history options,
we used multiplex real-time PCR to assess the expression of 107 microRNAs throughout development in both continuous and
interrupted life histories. We identified 17 microRNAs whose developmental profile of expression is affected by dauer life
history and/or L1 diapause, compared to continuous development. Hence these microRNAs could function to regulate gene
expression programs appropriate for different life history options in the developing worm.
Keywords: microRNA; dauer; development

INTRODUCTION late translation and mRNA stability through a variety of

mechanisms, depending on cellular context and environ-
Animal development is a complex, highly regulated pro-
mental conditions (Hammell 2008; Holtz and Pasquinelli
cess, whose outcome is vital to the survival of the individual
2009). Because a given miRNA can regulate the expression
and the species. Animals in their natural environments can
of multiple target genes, miRNAs can potentially provide
develop normally despite varying and harsh environmental
coordinated rapid changes in gene expression in response
conditions. Hence, animals have evolved mechanisms to
to developmental, environmental, and physiological cues
ensure that developmental outcomes occur robustly, regard-
(Bartel 2004, 2009). Indeed, the expression of many
less of changing conditions. MicroRNAs (miRNAs) may play
miRNAs is developmentally regulated, consistent with roles
an important role in developmental robustness to changing
for miRNAs in the regulation of gene expression in con-
environments. These small, noncoding RNAs bind to par-
junction with developmental processes (Bartel 2004; Kato
tially complementary sequences in target mRNAs and regu-
and Slack 2008). Furthermore, roles for miRNAs in pro-
viding robustness of developmental outcomes to various
1
Present address: Columbia University Medical Center, HHMI/ perturbations have been demonstrated (Li et al. 2009;
Department of Biochemistry and Molecular Biophysics, New York, NY Hilgers et al. 2010; Rasmussen et al. 2010).
10032, USA. Caenorhabditis elegans is an excellent system for the study
2
Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY 11724, USA. of developmental robustness to environmental perturbations
Reprint requests to: Victor Ambros, Program in Molecular Medicine, because this animal possesses mechanisms to respond to
University of Massachusetts Medical School, Worcester, MA 01605, USA; fluctuations in environmental conditions and to adjust de-
e-mail: victor.ambros@umassmed.edu; fax: (508) 856-5657.
Article published online ahead of print. Article and publication date are velopmental progression accordingly, without compromis-
at http://www.rnajournal.org/cgi/doi/10.1261/rna.2310111. ing the integrity of cell fate specification events (Liu and

RNA (2011), 17:00–00. Published by Cold Spring Harbor Laboratory Press. Copyright ! 2011 RNA Society. 1
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Karp et al.

Ambros 1991; Euling and Ambros 1996; Riddle and Albert and the dauer larvae remain at a developmental stage equiv-
1997; Braendle and Félix 2008). In order to identify miRNAs alent to the L2-to-L3 molt (L2m) (Cassada and Russell
that could mediate interactions between physiological and 1975). When environmental conditions improve, dauer
developmental pathways, we assayed the expression level of larvae restart their cell division cycles and re-enter develop-
107 miRNAs during development through alternative life ment, progressing through post-dauer L3 and post-dauer L4
histories: the continuous development that occurs in favor- (PDL3, PDL4) stages (Riddle and Albert 1997). We refer
able environments, and the interrupted development that to these two alternative developmental paths—of either
can occur in unfavorable environments. continuous or dauer-interrupted development—as ‘‘life
C. elegans development consists of embryogenesis fol- histories’’ (Fig. 1; Fielenbach and Antebi 2008).
lowed by four larval stages (L1–L4) punctuated by molts Besides the dauer diapause, which occurs after the L2d
(see Fig. 1). In favorable environments, larval development stage, C. elegans larvae can undergo another, distinct dia-
proceeds rapidly and continuously over z40–50 h at 20°C pause at the beginning of L1. The L1 diapause occurs prior
(Sulston and Horvitz 1977). Unfavorable environmental to larval development, in situations in which embryos
conditions sensed during L1 cause the larva to progress to hatch in the absence of food (Fig. 1). Larvae in L1 diapause
the L2d stage, which is z50% greater in duration than the share some features with larvae in dauer diapause: They are
rapid L2 stage (Golden and Riddle 1984). L2d larvae pre- developmentally arrested and nonfeeding. Furthermore,
pare for future harsh conditions, while continuing to assess larvae in L1 diapause display some stress resistance and
environmental cues. If conditions remain poor, larvae will lengthened life span, although to a lesser extent than dauer
enter the dauer diapause, a developmentally arrested, long- larvae (Johnson et al. 1984). Upon exposure to food, these
lived, and highly stress-resistant stage (Riddle and Albert arrested larvae exit L1 diapause and proceed with larval
1997). All cells remain quiescent during dauer diapause, development. Post-diapause L1 animals are not necessarily
committed to L2d or dauer arrest; they can choose either
continuous or dauer-interrupted life histories depending
on the environmental conditions encountered as they prog-
ress through the first two larval stages (Golden and Riddle
1984; Johnson et al. 1984).
Since the pace and/or continuity of larval development
are profoundly affected by larval life history, while the
patterns of cell divisions and the associated sequences of
cell lineage fates are unaffected, there must be mechanisms
to integrate the progression of cell lineage development
with the environmental and physiological signals that govern
the dauer versus continuous life history choices. Genes
involved in enabling animals to successfully traverse these
distinct alternative life histories could include genes that
display life history–dependent differences in expression.
There is evidence that life history affects the expression
of protein-coding genes. The majority of previously pub-
lished studies have compared mRNA levels or protein levels
in dauer larvae to non-dauer animals of various develop-
mental stages (Dalley and Golomb 1992; Jones et al. 2001;
FIGURE 1. Schematic representation of continuous and interrupted
life history options. C. elegans larvae develop continuously through Holt and Riddle 2003; Wang and Kim 2003; Ruzanov et al.
four larval stages in favorable environmental conditions, but can 2007; Jeong et al. 2009b; Ruzanov and Riddle 2010). An
interrupt their development by entry to the stress-resistant L1 dia- additional study monitored gene expression changes during
pause or dauer diapause in unfavorable environmental conditions. entry to dauer (Jeong et al. 2009a). These studies focused
(Red shading) Unfavorable or (blue shading) favorable environmen-
tal conditions sensed by larvae. These environmental conditions on identifying genes that could contribute to the stress
drive larvae to either continuous or diapause-interrupted life histories. resistance, altered metabolism, and/or long life span char-
(Dashed lines) Junctures where larvae may switch between diapause- acteristic of dauer larvae, rather than on the developmental
interrupted and continuous life history depending on the severity of effects of dauer diapause. However, two previous studies
the conditions encountered. Developmental stages at which miRNA
levels were assessed in each life history: (blue) stages are within the did compare mRNA levels between developmentally equiv-
continuous life history; (red) stages are within the dauer-interrupted alent stages in different life histories. Liu et al. (2004)
life history. Developmentally equivalent stages were compared (Table compared late L2d/early dauer TGFb mutants to late L2/
1); these are the red and blue stages at the same point along the
vertical axis. (Orange) L1 diapause, which can lead to either con-
early L3 staged wild-type larvae, and Harvey et al. (2009)
tinuous or dauer life histories, was compared to both embryos and compared wild-type early-staged L2d larvae to wild-type
continuously developing L1 larvae (Table 2). continuously developing L2 larvae. Liu et al. (2004) found

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Dauer affects miRNA expression during development

changes in the expression of many genes that regulate dauer RESULTS

formation, and also in several developmental regulators
such as Notch pathway genes, Hedgehog pathway genes, The expression of 14 miRNAs is affected by dauer
and genes that regulate stage-specific cell fate decisions. life history
Some of these changes could be part of the coordination
between pathways that regulate development and pathways To ascertain the expression of most (107) C. elegans
that regulate life history choice. miRNAs during development through both continuous
Less is known about gene expression changes in post- and dauer-associated life histories, we isolated RNA from
dauer larval stages. However, a recent study compared animals grown at 20°C in either favorable or dauer-pro-
wild-type young adult hermaphrodites that had developed moting environmental conditions. To collect staged larvae
either continuously or whose development was interrupted developing continuously in favorable environmental con-
by dauer (Hall et al. 2010). The investigators found not ditions, embryos were isolated by hypochlorite treatment of
only numerous mRNAs whose levels were altered, but also eggs and gravid adults. These embryos were added to plates
global chromatin-remodeling signatures in post-dauer containing plenty of food and space. Larvae grew at sparse
adults. Furthermore, they found that the post-dauer chro- population density until the appropriate stage (L1, L2,
matin state was associated with increased brood size, L2m, L3, or L4), as determined developmentally (see
suggesting that the epigenetic changes that occur in the Supplemental Methods). To collect staged L1 and L2 larvae
dauer life history can affect the phenotype of wild-type that would have been likely to enter dauer diapause had they
individuals. It is not known whether transcription of miRNA- not been harvested, embryos were added to growth medium
encoding genes is similarly affected by these chromatin containing dauer pheromone, a potent environmental cue
modifications. for eliciting L1d, L2d, and dauer formation (Golden and
Previous work has identified certain miRNAs whose Riddle 1982; Butcher et al. 2007). Larvae were grown in
developmental expression is affected by life history. We parallel with or without pheromone, and larvae from each
and others have shown that let-7-family miRNA expression culture were harvested for RNA extraction at equivalent
is reduced in L2d larvae and in dauer larvae by the re- developmental stages. Dauer larvae were isolated by SDS
pressive activity of the DAF-12 nuclear hormone receptor selection from plates that had a high population density and
(Bethke et al. 2009; Hammell et al. 2009). This repression is on which animals had exhausted their food supply. For
part of a feedback loop that functions to integrate stage- comparison to dauer larvae, animals that had developed
specific cell fate decisions with life history choice (Hammell continuously in favorable environmental conditions were
et al. 2009). In our previous study (Hammell et al. 2009), harvested at the L2 molt and early L3 (see Fig. 1). Similarly,
we compared the expression of 107 miRNAs during con- post-dauer L3 (PDL3) and post-dauer L4 (PDL4) larvae
tinuous L2 and L2d. We found four let-7-family miRNAs were obtained by selecting dauer larvae as above, then adding
and one additional miRNA, miR-788, to be down-regulated the dauer larvae to plates with abundant food to support the
in L2d. In a separate report, miR-788 was shown to be resumption of development. PDL3 and PDL4 larvae were
down-regulated during dauer diapause, likely by direct harvested when they reached a stage of development equiv-
action of the DAF-3 transcription factor (Martinez et al. alent to the continuously developing L3 and L4 larvae, re-
2008a). In addition, both miR-248 and miR-234 were spectively. For each stage and life history, three independent
found to be up-regulated during dauer diapause by un- biological replicates were obtained in this manner.
known mechanisms (Lim et al. 2003). There are numerous methods for quantifying miRNA
In the present study, we expand on those previous levels including mir-Taqman real-time PCR, Northern blot,
studies and present a comprehensive profiling of 107 deep sequencing, microarrays, and reporter transgenes. All
worm miRNAs across the entirety of larval development— have some benefits and some caveats associated with them
including animals in L1 diapause, animals at all stages of (Git et al. 2010). We chose the multiplex miR-Taqman
continuous development, and animals at all stages of assay to quantify miRNA levels because it is highly quan-
dauer-associated life histories. We find 14 miRNAs whose titative, can discriminate between miRNA family members,
expression is affected by dauer life history during at least can measure levels of multiple miRNAs simultaneously,
one larval stage. In addition, we identify five miRNAs that and measures the endogenous, mature miRNA species.
are up-regulated during L1 diapause, two of which are also Furthermore, a very small amount of RNA is required, per-
up-regulated in the dauer life history. We discuss these mitting us to grow relatively small numbers of animals in
effects of life history on miRNA expression in the context specific environmental conditions. To verify the specificity of
of overall development by comparing the trajectory of their this assay, we tested strains lacking eight different miRNAs
expression across all larval stages in both life histories. within the let-7 and miR-35 families. None of the miRNAs
These findings serve as the basis for forming hypotheses that were genetically deleted were detectable in this assay,
about the function of miRNA regulatory mechanisms in life whereas wild-type miRNAs were readily detectable (Supple-
history choices and stress resistance. mental Table S1).

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Karp et al.

To ascertain which miRNAs differed significantly be- appear in Table 1 are either poorly expressed (Supplemen-
tween the life histories, we obtained the mean Ct values for tal Table S2), variable between experiments (Supplemental
each stage and life history across all three biological rep- Table S4), or more ambiguous with respect to fold-change
licates. Mean Ct values were normalized to the 25 micro- between life histories, such that they do not meet the
RNAs that were least variant across all stages and condi- criteria we define above (Supplemental Table S3).
tions. The complete list of these normalized mean Ct values We noticed that miRNAs whose expression changes
and the standard deviations associated with them are listed dramatically during a particular larval stage displayed a high
in Supplemental Table S2. We observed a range of ex- standard deviation at that stage. For example, lin-4 expres-
pression levels for the microRNAs that we assayed, and sion is very low in newly hatched L1s but high by the end of
a range of developmental profiles (Fig. 2). L1 (Feinbaum and Ambros 1999). We find a high standard
The normalized mean Ct values and their standard deviation for the lin-4 Ct values of the three biological
deviations were used to calculate fold-changes, error, and replicates during the L1 stage, but not other stages when
statistical significance between developmentally equivalent lin-4 levels are more stable (Supplemental Table S2). This is
stages of continuous versus dauer-associated development likely due to the fact that each biological replicate captures
(see Fig. 1). These calculations were carried out by standard an imperfectly synchronized population of larvae (see
methods (see Materials and Methods; AppliedBiosystems Supplemental Methods).
2008). Fold-changes were deemed significant if they met all
of the following criteria: (1) The fold-change had a P-value
Five miRNAs are significantly up-regulated
of #0.05 as assessed by t-test (Materials and Methods); (2)
during L1 diapause
the fold-change was $twofold; and (3) the miRNA was
expressed highly enough to be well measured by the assay In order to identify miRNAs whose expression is affected
(Supplemental Methods). The complete list of fold-changes by L1 diapause, we compared starved L1 (stL1) larvae
of all 107 miRNAs at each larval stage is provided in (starvation of newly hatched L1 larvae causes them to arrest
Supplemental Table S3. To assess the reproducibility of in L1 diapause) to embryos and to continuously developing
these results, particularly during dauer diapause when gene L1 larvae. We define miRNAs whose expression responds
expression in general is substantially affected, we performed to L1 diapause to be those whose expression is higher (or
completely independent experiments, assaying all miRNA lower) in stL1 animals compared to both embryo and L1
levels in samples of staged populations of dauer and L3 stages. From this comparison, we identified five miRNAs
larvae obtained independently of those in Supplemental that were significantly up-regulated in stL1 larvae (Table 2),
Tables S2 and S3. The results of these experiments were and no miRNAs to be significantly down-regulated in stL1
essentially in agreement with those in Supplemental Table larvae (Supplemental Table S6).
S3. There were a few cases of discordant results, in which StL1 larvae share some features with dauer larvae: They
an miRNA displayed a significant change in dauer larvae in are developmentally arrested, quiescent, and nonfeeding.
one set of experiments, but not the other set. The data for However, unlike dauer larvae, stL1 larvae are active in
these are listed in Supplemental Table S4. Only those pharyngeal pumping, do not adopt the dauer-characteristic
miRNAs for which the results of both sets of experiments cuticle morphology, and lack the extreme longevity and
were in agreement are reported in Table 1 and Figure 3. stress resistance of dauer larvae (Johnson et al. 1984). The
Using the above criteria, we identified 14 miRNAs that five miRNAs we find to be significantly up-regulated in stL1
were significantly different in level when corresponding larvae show a range of behaviors in the dauer larva life
stages of the continuous and dauer life histories are com- history. miR-238 is unaffected by dauer life history at any
pared (for at least one stage) (Fig. 3; Table 1). In Supple- stage (Table 1), suggesting that its up-regulation in stL1
mental Table S5, we also provide a list of 15 miRNAs whose larvae is a specific response to L1 diapause. In contrast,
expression changes are less substantial (1.5–1.9-fold), yet still miR-34 and miR-71 are also up-regulated in L2d, dauer,
statistically significant by t-test (Materials and Methods). In and PDL3 stages (Fig. 3; Table 1), suggesting that up-
our analysis, however, we focus on miRNAs whose levels regulation of these miRNAs may be a more general response
were changed at least twofold, because we have greater to environmental stress. miR-234 and miR-254 are both up-
confidence in the biological relevance of expression changes regulated in dauer diapause (P # 0.05), but the fold-change
of this magnitude. is <2, such that we have less confidence in the biological
In contrast to the miRNAs that we found to be sig- significance of this effect (Supplemental Table S5).
nificantly affected by life history, we also identified a set of
miRNAs that, based on our data, appear to be unaffected by
Developmental profiles of miRNA expression
life history (Table 1). These miRNAs are well expressed, as
defined by having Ct values <30; their mean fold-change To characterize how the developmental trajectories of
across the three biological replicates was #1.4–1.5, and the miRNA expression are affected by life history, we per-
error was #0.53 the fold-change. miRNAs that do not formed hierarchical clustering on the Ct values of all 107

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Dauer affects miRNA expression during development

FIGURE 2. Hierarchical clustering of miRNA expression profiles during developmental progression. Hierarchical clustering was carried out
using Cluster 3.0 and visualized using Java TreeView programs (Materials and Methods) on (A) embryo (Emb), L1, L2, L3, and L4 staged
continuously developing larvae or (B) embryo (Emb), L1d, L2d, PDL3, and PDL4 staged larvae progressing through the dauer life history. Note
that a 1-Ct difference corresponds to a twofold change in expression level. (**) An miRNA whose expression changes significantly and
substantially between life histories (see Fig. 3; Table 1).

miRNAs in dauer versus continuous life histories. In order diapause and dauer diapause were omitted) (Fig. 2). The
to focus on progression through development, we used clusters we observed were generally similar between life
only stages that were not developmentally arrested (i.e., L1 histories (Fig. 2). In each case, hierarchical clustering

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Karp et al.

TABLE 1. The effect of the dauer life history on miRNA expression

miR-35 family miRNAs are highest in
embryos and decline thereafter (Abbott
Up-regulateda Down-regulateda Unaffectedb et al. 2005; Esquela-Kerscher et al. 2005;
Familyc microRNA Stage Familyc microRNA Stage Familyc microRNA Kato et al. 2009a). To better quantify
how our data for miRNA expression
miR-34 miR-34 L2, D, L3 let-7 miR-48 L3 miR-44 miR-44
correspond to previously published data,
miR-84 D miR-45
Otherc miR-71 D, L3 miR-241 D, L3 we compared our Ct values to the
miR-248 D, L3 miR-795 L1, D miR-49 miR-83 number of sequence reads in a deep-
lin-4 miR-237 L1, D, L4 miR-51 miR-52 sequencing analysis performed in em-
miR-53 bryo, L1, L2, L3, and L4 stages (Kato
miR-1 miR-1 L3 miR-54
et al. 2009a). At each of these develop-
miR-56
miR-2 miR-250 D mental stages, we find that our data
miR-57 miR-57 correspond well to the deep-sequencing
Other miR-70 L3 data for z75% of the miRNAs (Supple-
miR-230 D miR-58 miR-58 mental Table S7). The 25% of miRNAs
miR-788 D, L3
with poor agreement between the two
miR-799 D miR-63 miR-66
miR-288 data sets could reflect various factors
including (1) variability in miRNA ex-
miR-72 miR-73 pression during a stage when it is being
up- or down-regulated, (2) different se-
miR-86 miR-8
quence biases inherent to the mirTaqman
miR-87 miR-87 and deep sequencing assays (Willenbrock
miR-233 et al. 2009; Git et al. 2010), and (3)
differences in growth conditions in the
miR-238 miR-238 two studies (Ruzanov and Riddle 2010;
for more details, see Supplemental Me-
other miR-60
thods and Supplemental Table S8).
a
miRNAs whose expression is significantly (P # 0.05, by t-test) and substantially ($twofold)
affected by life history, when corresponding developmental stages are compared. The
developmental stages substantially and significantly affected are indicated; (L1) L1 versus Developmental trajectory
L1d; (L2) L2 versus L2d; (D) L2 molt versus dauer; (L3) L3 versus PDL3; (L4) L4 versus PDL4.
‘‘Up-regulated’’ indicates that levels of that miRNA are higher in the dauer life history;
of miRNAs significantly
‘‘down-regulated’’ indicates that levels are lower in the dauer life history. See Supplemental affected by life history
Table S5 for a list of miRNAs whose expression is affected significantly but not substantially
(1.5–1.9-fold), Supplemental Table S3 for a complete list of the effect of life history on allTo ascertain how developmental pro-
107 miRNAs assayed, and Supplemental Table S4 for a list of miRNAs affected differently in files of miRNA expression may be af-
independent experiments.
b
High confidence unaffected miRNAs meet the following criteria: miRNAs were well-
fected by life history, we conducted a
measured (Ct’s were <30 and the error was <0.53 the fold-change), and the fold-change side-by-side comparison of correspond-
between life histories was <1.43 across at least four larval stages, and <1.53 in the ing stages of dauer versus continuous
remaining larval stage.
c
Family members were defined as Ibáñez-Ventoso et al. (2008); ‘‘other’’ indicates microRNAs
life histories for the 14 miRNAs whose
not assigned in a family. expression is significantly affected by
life history at one or more larval stage
(Fig. 3). In this comparison, we in-
produced two major groups of miRNAs: those with low to cluded expression levels for the L1 diapause and dauer
moderate expression and those with high expression. As larva stages. We find four patterns of effect on the
subclasses within these groups we find (1) developmentally trajectory of miRNA expression. First, the developmen-
regulated clusters of miRNAs—those whose expression tal up-regulation of lin-4 and let-7-family microRNAs is
dramatically increases or decreases during progression delayed in the dauer life history (Fig. 3A). The dramatic up-
through development; (2) developmentally uniform clus- regulation of miR-237, miR-48, miR-84, and miR-241
ters of miRNAs; and (3) a small set of outliers whose observed during continuous development is consistent with
expression changes in different directions across develop- previous reports (Abbott et al. 2005; Esquela-Kerscher et al.
ment (Fig. 2). 2005). The delay in up-regulation of let-7-family micro-
The developmental profiles of miRNA expression that we RNAs that we observe throughout the dauer life history
observe for continuous development are generally consis- (Fig. 3A) is consistent with the model that unfavorable
tent with previously published developmental profiles. For environmental conditions cause the repressive form of
example, we find lin-4-family and let-7-family miRNAs to DAF-12 to dampen the intrinsic up-regulation of let-
be up-regulated during development, whereas the levels of 7-family miRNAs as part of a feedback loop that coordinates

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Dauer affects miRNA expression during development

in the dauer life history (Fig. 3B). Indeed,

miR-788 clusters with developmentally
up-regulated miRNAs in continuous de-
velopment, but with developmentally
uniform miRNAs in the dauer life his-
tory (Fig. 2).
Third, the trajectory of three
miRNAs—miR-34, miR-71, and miR-
248—is shifted from relatively uniform
during continuous larval development
to dynamic expression during develop-
ment through the dauer life history
(Fig. 3C). These three miRNAs are all
up-regulated in the dauer life history
(Table 1). Interestingly, the levels of
miR-34, miR-71, and miR-248 are also
increased in stL1 larvae relative to em-
bryos and L1 larvae (Fig. 3C), suggest-
ing that these miRNAs are generally
activated during periods of environ-
mental stress. This increase is substan-
tial (twofold to 23-fold) for all three
miRNAs, but statistically significant for
only miR-34 and miR-71 (Table 2; Sup-
plemental Table S6). The substantial
change in the developmental trajectory
of these miRNAs suggests that their
targets might need to be down-regulated
during a large portion of progression
FIGURE 3. Developmental trajectory of miRNAs whose expression is significantly different through the dauer life history.
between life histories. Four different effects on developmental trajectory were observed (A-D). Finally, for five additional miRNAs,
See text for details. The expression of miRNAs whose expression is substantially and signifi-
cantly affected at one or more larval stages (Table 1) was represented by the same color-coding
the developmental trajectories are sim-
scheme as in Figure 2. To directly compare progression through continuous and dauer- ilar in continuous and dauer life histo-
interrupted life histories, the corresponding developmental stages are shown in parallel, with ries, with the exception of a single stage
the continuous life history on the top and the dauer life history on the bottom. Developmental at which they are significantly affected
diapauses are also shown, including L1 diapause (stL1) and dauer diapause (D). Note that
embryo and stL1 stages can lead to either continuous or dauer life history. Numbers within
in the dauer life history (Fig. 3D).
squares indicate the fold-change in expression between continuous and dauer life histories
(continuous/dauer). Note that in some cases, an approximately twofold change in expression
does not cause a change in the color, or conversely that a small change in Ct can cause a color Life history can affect the
change if the Ct was near the boundary defined. Also note that a 1-Ct difference is a twofold transcription of miRNAs
change in miRNA level. See Supplemental Table S3 for a complete list of the fold-change in
miRNA expression between different life histories at all larval stages for all 107 miRNAs, Previously published reports have dem-
including the error, and P-value, Supplemental Table S5 for a list of miRNAs that change onstrated transcriptional down-regulation
significantly but not substantially (1.5–1.9-fold), and Supplemental Table S4 for a list of miRNAs of several miRNAs during dauer dia-
that are affected differently in independent experiments. (*) A statistically significant fold-change
is observed when the miRNA level in stL1 is compared to the miRNA level in both embryo and pause. These include the let-7-family
continuously developing L1 stages (see Table 2). (y) The substantial fold-change observed at this miRNAs that are direct targets of the
stage is not statistically significant in this analysis (Supplemental Table S3). These miRNAs are DAF-12 nuclear hormone receptor,
dramatically up-regulated in L2 staged larvae (Supplemental Table S2; Abbott et al. 2005), causing
and miR-788, which is a direct target
any small variation in the stage of each sample to lead to a large variation in the amount of miRNA
present in different biological replicates. However, when these same samples were compared using of DAF-3/Smad (Martinez et al. 2008a;
Bethke et al. 2009). We therefore won-
methods to assess fold-change for cultures grown side by side, we did find a statistically significant
difference at this stage (Hammell et al. 2009; Pradervand et al. 2009). dered whether the effect of life history
on the levels of other miRNAs might be
developmental timing with life history choice (Hammell mediated transcriptionally. Since dauer diapause is the stage
et al. 2009). at which life history appears to exert the greatest effect on
Second, the developmental up-regulation of miR-788 the levels of mature miRNAs (Fig. 3), we focused on the
that occurs during continuous development was abolished comparison between dauer diapause and the corresponding

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Karp et al.

TABLE 2. miRNAs increased in starvation-induced L1 diapause

of overlap of terms with significant enrichment (Table 3).
(stL1), compared to embryos and developing L1 larvae Exceptions include kinase-related functions enriched among
predicted targets of miRNAs down-regulated in the dauer
miRNA Fold-change stL1/Emb Fold-change stL1/L1
life history, and several primarily molecular categories
miR-34 23.2 6 12.6 5.8 6 1/2
miR-71 16.8 6 6.7 2.2 6 0.9
miR-234 3.6 6 1.2 2.6 6 0.6
miR-238 9.1 6 3.5 3.6 6 1.4
miR-254 2.4 6 0.5 3.1 6 1.2

P < 0.05 for both stL1/Emb and stL1/L1.

stage of continuous development (approximately the L2

molt and early L3 stage). We examined the expression of
transcriptional reporters for the three miRNAs whose levels
are most affected by dauer diapause, aside from the above-
mentioned let-7 family and miR-788 (Fig. 3; Supplemental
Table S3). These miRNAs are miR-34 (sixfold up-regu-
lated), miR-237 (sevenfold down-regulated), and miR-230
(14-fold down-regulated). All three of these reporters
exhibited changes in expression consistent with our obser-
vations of the mature levels (Fig. 4). Specifically, levels of
mir-237TGFP and mir-230TGFP were reduced in dauer
larvae, whereas levels of mir-34TGFP were increased in
dauer larvae (Fig. 4). Therefore, the effect of life history on
the levels of these particular miRNAs is mediated in large
part transcriptionally.

Functional terms enriched among predicted targets

of miRNAs whose expression is affected by life history
As genetic studies of miRNA mutants are only beginning to
reveal the function of most miRNAs in C. elegans (Miska
et al. 2007; Alvarez-Saavedra and Horvitz 2010; Brenner
et al. 2010), we estimated the types of biological processes
that might be affected by life history as a result of changes
in miRNA levels. First, miRNAs were placed into one of
four groups: up-regulated during L1 diapause, up-regulated
in the dauer life history, down-regulated in the dauer life
history, or unaffected by dauer life history (Tables 1, 2). FIGURE 4. Transcriptional regulation of miRNA expression in dauer
Next, predicted targets of miRNA families within each diapause. Expression of transcriptional reporters of miRNA expres-
sion during continuous development (L2 molt or early L3 stage, prior
group were identified using mirWIP (Supplemental Table to VPC division) or during dauer diapause. (A–F) Expression of a
S9; Hammell et al. 2008). Finally, for each group of pre- mir-237TGFP reporter (Martinez et al. 2008b). (A and D.) GFP
dicted targets, significantly enriched functional terms were fluorescence observed at a 200-msec exposure time using a 103
identified using DAVID (Huang et al. 2009). objective. (B,E) Magnification of the boxed region in A and D.
Fluorescence observed at a 19-msec exposure time using a 403
We find that predicted targets of miRNAs up-regulated objective. (C,F) DIC images corresponding to B, E, respectively. (G–L)
in the dauer life history are enriched for developmental Expression of a mir-230TGFP reporter (Martinez et al. 2008b). (G,J)
proteins and alternative splicing-related functions (Table GFP fluorescence observed at a 100-msec exposure time using a 103
3). Since alternative splicing is an important aspect of the objective. (H,K) Magnification of the boxed region in G and J,
respectively. Fluorescence observed at a 3-msec exposure time using
developmental control of gene expression in C. elegans, the a 403 objective. (I,L) DIC images corresponding to H and K, re-
predicted down-regulation of these genes is consistent with spectively. (M–R) Expression of an mir-34TGFP reporter containing
the interruption of development within the dauer life 5 kb of promoter sequence. (M,P) GFP fluorescence observed at a
200-msec exposure time using a 103 objective. (N,Q) Magnification
history (Zahler 2005; Rukov et al. 2007). These terms were of the boxed region in M and P, respectively. Fluorescence observed
not enriched within other groups (Table 3). In contrast, pre- at a 50-msec exposure time using a 403 objective. (O,R) DIC images
dicted targets of the other three groups show a high degree corresponding to N and Q, respectively.

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Dauer affects miRNA expression during development

TABLE 3. Significantly enriched functional terms among predicted miRNA targets

that occur in their natural environment.
The importance of the ability of C.
stL1a Upb Downc Unaffectedd elegans to choose the dauer life history
Term False discovery rate e is underscored by the observation that
samples of C. elegans isolated from the
Alternative splicing — 0.0002 — —
wild are most often found in the dauer
Splice variant — 0.04 — —
Developmental protein — 0.01 — — stage (Barriere and Felix 2005). This
ATP-binding — — 0.006 — choice between continuous and dauer-
Serine/threonine-protein kinase — — 0.02 — interrupted life histories involves dra-
Nucleotide-binding 0.006 — 0.0002 — matic effects on developmental progres-
GO:0032504;multicellular organism
sion, including a lengthening of pre-
reproduction 0.01 — 0.002 0.002
GO:0048609;reproductive process in a dauer stages and the postponement of
multicellular organism 0.01 — 0.002 0.002 post-dauer stages (Golden and Riddle
Nucleus 0.03 — 0.02 0.02 1984). These processes certainly involve
IPR007087:Zinc finger, C2H2-type — — 0.03 0.01 widespread and coordinated regulation
GO:0018991;oviposition 0.03 — — 0.0002
of gene expression, on the transcrip-
GO:0033057;reproductive behavior in a
multicellular organism 0.04 — — 0.0003 tional and post-transcriptional levels.
GO:0019098;reproductive behavior 0.05 — — 0.0004 Changes in gene transcript levels have
GO:0006928;cell motion — — — 0.003 been documented that accompany the
GO:0007610;behavior — — — 0.002 increased stress resistance, reduced me-
IPR003598:Immunoglobulin subtype 2 — — — 0.006
tabolism, and lengthened life span char-
IPR003599:Immunoglobulin subtype — — — 0.02
IPR007110:Immunoglobulin-like — — — 0.02 acteristic of dauer larvae (Dalley and
DNA-binding — — — 0.01 Golomb 1992; Jones et al. 2001; Holt
Kinase — — — 3.5E-06 and Riddle 2003; Wang and Kim 2003;
Transmembrane — — — 0.02 Ruzanov et al. 2007; Jeong et al. 2009b;
IPR011991:Winged helix repressor
Ruzanov and Riddle 2010). Similarly,
DNA-binding — — — 0.004
IPR013098:Immunoglobulin I-set — — — 0.001 transcriptional changes have been de-
IPR015880:Zinc finger, C2H2-like — — — 0.007 scribed associated with L1 diapause
(Baugh et al. 2009). Less is known about
Significantly enriched functional terms of predicted targets of miRNAs, compared with the
the contribution of post-transcriptional
complete set of annotated transcripts. MiRNAs were scored as follows: regulation of gene expression in the
a
Up-regulated in L1 diapause (‘‘stL1’’). choice between continuous and inter-
b
Up-regulated in the dauer life history.
c
Down-regulated in the dauer life history.
rupted life histories. microRNAs are
d
Unaffected by life history (Tables 1, 2). Predicted targets are listed in Supplemental Table candidates for acting post-transcrip-
S9. tionally to coordinate programs of dif-
e
False discovery rate as supplied by DAVID (Huang et al. 2009). Note that Bonferroni and
Benjamini adjusted P-values were also <0.05 in all cases where the false discovery rate was
ferential gene expression, as individual
<0.05. Dashes indicate a false discovery rate $0.05. microRNAs have the potential to regu-
late multiple target genes (Bartel 2004,
2009). We present a quantitative assess-
enriched among predicted targets of miRNAs that are ment of miRNA expression throughout continuous and
unaffected by life history (Table 3). Interestingly, we did diapause-interrupted life histories that provides a valuable
not find dauer-related terms to be enriched in any group. resource to researchers studying any aspect of development
We did find three important genes that regulate dauer that involves L1 diapause, dauer diapause, or other aspects
formation—daf-5, daf-12, and daf-16—to be predicted of dauer-associated life history.
targets of several miRNAs identified in this study (Supple-
mental Table S9). However, because duplicate entries are
Biological processes that may be affected by altered
not included in the analysis, in order for a functional term
miRNA levels in different life histories
to be enriched there would need to be many different
predicted targets that have been assigned that term, rather We hypothesize that miRNAs may be involved in in-
than a few key genes appearing multiple times. tegrating the developmental and physiological effects of
life history choice. Consistent with this hypothesis, we find
that miRNAs that are affected by life history are also likely
DISCUSSION
to fall into developmentally regulated clusters in the
The stress-resistant dauer diapause permits C. elegans continuous life history (seven of 14 miRNAs) (Fig. 2A),
larvae to withstand the harsh environmental conditions whereas miRNAs that are unaffected by life history are

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Karp et al.

likely to fall into developmentally uniform clusters (13 of miRNAs whose expression is affected by life history
17 miRNAs) (Fig. 2A). This general trend suggests a re- (Esquela-Kerscher et al. 2005; Martinez et al. 2008b; Kato
lationship between developmental progression and the et al. 2009b; Alvarez-Saavedra and Horvitz 2010). These
physiological changes that accompany life history choice, miRNA gene transcriptional reporters showed various
such that the targets of miRNAs that are present at expression patterns, including broad temporal and ana-
relatively uniform levels throughout development may tomical expression and more specific expression patterns.
remain at those same levels in either life history. The three tissues where reporter expression was most often
A relationship between gene expression changes in dauer seen were the hypodermis, the vulva, and the nervous
larvae and gene expression changes that occur in aging system (Supplemental Table S10). The hypodermis is an
adults has been described (for review, see Golden and endocrine tissue where signaling regulating the dauer
Melov 2007). Indeed, many of the same genes that regulate formation decision occurs (Gerisch and Antebi 2004).
dauer formation also regulate life span (see Golden and Furthermore, the timing of stage-specific cell fate decisions
Melov 2007; Hu 2007). Two previous studies characterized within the hypodermis is regulated by lin-4 and let-
the expression of miRNAs during aging in wild-type adult 7-family miRNAs. This regulation can be altered by dauer
C. elegans hermaphrodites (Ibáñez-Ventoso et al. 2006; de formation (Liu and Ambros 1991). The vulva is an
Lencastre et al. 2010). While many of the miRNAs whose ectodermal tissue that is lineally related to some hypoder-
expression is affected by aging are distinct from those mal cells (Sulston and Horvitz 1977). It has been noted that
whose expression is affected by life history, it is interesting mutations in genes affecting vulval development can pro-
to note that miR-34 and miR-71, two of the three miRNAs duce altered phenotypes when these animals progress
that are up-regulated in the dauer life history (Table 1), are through dauer life history (Ferguson and Horvitz 1985;
also up-regulated in older adults (Ibáñez-Ventoso et al. 2006; Euling and Ambros 1996; Braendle and Félix 2008). Finally,
de Lencastre et al. 2010). Intriguingly, miR-71, promotes certain sensory neurons are the source of signals regulating
longevity, as well as resistance to both heat and oxidative the dauer formation decision. Furthermore, the morphol-
stress in adulthood, whereas miR-34, is required for a robust ogy of some neurons is altered in dauer diapause (for
DNA damage response (Kato et al. 2009b; de Lencastre et al. review, see Riddle and Albert 1997; Hu 2007). Therefore,
2010). These roles are consistent with the up-regulation of many of the miRNAs whose levels are affected by life
these miRNAs during L1 diapause and in the dauer life history are apparently transcribed in tissues that are also
history, in response to environmental stress (Fig. 3). affected by life history and/or that participate in life history
choices.
In conclusion, we have identified a core set of miRNAs
Transcriptional regulation of miRNA expression
whose expression is altered by diapause-associated life his-
The data presented in Figure 4, combined with previous tory. These microRNA include four let-7-family microRNAs.
reports, demonstrate that the effect of dauer diapause The let-7 family had been previously implicated in a mech-
on miRNA expression can be mediated transcriptionally anism that coordinates stage-specific cell fate decisions with
(Martinez et al. 2008a; Bethke et al. 2009). The previously life history choice (Hammell et al. 2009). This coordination
characterized transcriptional regulation was assessed by the is necessary for the robustness of cell fate specification to
association of core dauer pathway transcriptional regula- environmental changes and life history that is observed in
tors with miRNA promoters. These core components wild-type C. elegans animals (Liu and Ambros 1991; Euling
include the DAF-12 nuclear hormone receptor, which is and Ambros 1996; Braendle and Félix 2008). We have also
absolutely required for dauer formation, and the DAF-3 identified 10 other microRNAs whose expression is altered
Smad, which promotes dauer formation downstream from by life history choice. Future genetic analysis of the func-
TGF-b signaling (Thomas et al. 1993; Patterson et al. 1997; tions of these additional miRNAs should reveal whether
Antebi et al. 2000). Yeast one-hybrid assays have been and how they may also participate in the coordination of
performed for the promoters of most miRNAs (Martinez developmental and physiological gene expression in the con-
et al. 2008a). Interestingly, DAF-3 binds to seven of the text of diapause-associated life history choices.
nine tested promoters of miRNAs whose mature levels are
affected by life history. In contrast, DAF-3 binds to only
one of the 10 tested promoters of miRNAs whose expres- MATERIALS AND METHODS
sion is unaffected by life history (Supplemental Table S10;
Martinez et al. 2008a). This raises the possibility that the Strains and growth conditions
effect of life history on miRNA expression may be mediated The wild-type reference strain N2 was grown on the OP50
directly by the factors that regulate the life history choice bacterial strain at 20°C in all cases. To synchronize larvae, we
itself, at least in some cases. isolated embryos by hypochlorite treatment and then added them
Additionally, information about the expression of tran- directly to either standard NGM plates to prepare continuously
scriptional reporters is available for 10 of the promoters of developing larvae, or small (2 mL) NGM plates lacking peptone

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Dauer affects miRNA expression during development

and with the addition of 30 mg/mL streptomycin and 50-mL crude number of predicted targets of any of these miRNAs was 34. We
dauer pheromone (Vowels and Thomas 1994) to isolate L1d and therefore used the top 34 predicted targets of each of the miRNA
L2d larvae. Pheromone-containing plates were seeded with 20 mL families of the indicated group (up-regulated in L1 diapause, up-
of 63 OP50. To prepare dauer larvae and post-dauer stages, regulated in the dauer life history, down-regulated in the dauer life
a mixed-stage population of animals was grown on standard history, or unaffected by dauer life history) to compare to the
NGM plates until the food supply was exhausted and the entire C. elegans genome using functional annotation clustering
population density was sufficient to induce dauer larvae forma- within DAVID (Huang et al. 2009) in order to ask for significantly
tion. Dauer larvae were then selected by washing with 1% SDS and enriched functional terms.
either harvested immediately (for dauer larvae) or added to fresh
NGM plates seeded with OP50 (for post-dauer stages). All stages Hierarchical clustering
were confirmed by microscopic examination of stage-specific
anatomical characteristics, with the exception of the dauer stage, To compare normalized expression levels of miRNAs during
which was defined by SDS resistance. For more details, see the developmental progression, we used Cluster 3.0 to analyze !DCt
Supplemental Methods. values using an uncentered correlation and average linkage (de
Strains containing transcriptional reporters of miRNAs include Hoon et al. 2004). The DCt is the mean Ct of the three biological
VT1113 unc-119(ed3)III; maIs135[pmir-237TGFP, unc-119+], VL347 replicates, minus the mean Ct of the 25 least variant miRNAs (see
unc-119(ed3)III; wwEx19[pmir-230TGFP, unc-119+] (Martinez et al. the Supplemental Methods). We used the negative of the DCt so
2008b), and VT2514 unc-119(ed3)III; maEx221[pmir-345kbTGFP, that higher Ct values would be visualized as lower expression. The
unc-119+]. These strains were grown on 60-mm NGM plates seeded mean Ct of the 25 least variant miRNAs was 20.4 when nor-
with OP50 at 20°C. Dauer larvae were isolated from crowded and malized; therefore miRNAs with Ct values close to 20.4 had
starved plates and examined in parallel with continuously grown a !DCt of 0. The clustering was visualized with Java Tree View
larvae at the L2m or early L3 stage. Dauer larvae were picked from 1.1.3. Because the range of !DCt values displayed by this program
starved plates and examined directly (Fig. 4). The presence of dauer was limited to !3 to +3 (corresponding to normalized Ct values
alae was verified in these larvae. Images were obtained on a Zeiss of 17.4 to 23.4), we added additional colors using Adobe
Axio Imager D1 with an AxioCam MRm camera, and a X-Cite 120Q Illustrator to better represent miRNA expression outside this
light source (EXFO Photonic Solutions, Inc.). range. For clarity, the key shown in Figures 1 to 3 is in terms of
normalized Ct values, instead of !DCt values.
RNA preparation and mir-Taqman assay
Harvested larvae were snap-frozen in liquid nitrogen. RNA was SUPPLEMENTAL MATERIAL
extracted using the Trizol reagent (Invitrogen). Dauer larvae Supplemental material is available for this article, including a
(which have very tough cuticles) were subjected to three or more Supplemental Methods section (with additional experimental
rounds of freeze-cracking by alternating between liquid nitrogen details) and 10 Supplemental Tables of data, as referred to in the
and a 37°C water bath. Two microliters of a 3 ng/mL RNA dilution Results and Discussion sections.
was used for multiplex miR-Taqman reactions according to the
manufacturer, with an ABI 7900HT Fast-Real Time PCR System
(Applied Biosystems). ACKNOWLEDGMENTS
We thank Caifu Chen of Applied Biosystems Inc. for development
mir-Taqman analysis and use of the C. elegans–specific miR-TaqMan assays. We also
thank Iva Greenwald for the use of equipment and reagents. We
To identify the 25 least variant miRNAs across all conditions, we
are grateful to James Chen for advice about enrichment of
calculated the variance among the mean Ct’s of three biological
functional terms, and to Maria Sallee for helpful discussions and
replicates across all stages and conditions (embryo, stL1, L1, L2,
critical reading of the manuscript. This work was supported
L2m, L3, L4, L1d, L2d, dauer, PDL3, PDL4). The miRNAs
by National Institutes of Health Grants GM30428 (to V.A.),
identified are listed in Supplemental Table S2. For each larval
F32 GM73307 (to X.K.), F32 GM087039 (to M.H.), and F32
stage and life history, the mean of the 25 least variant miRNAs was
GM070118 (to M.C.O). Core resources supported by the Diabetes
calculated and then used to calculate DCt, DDCt, and fold-change
Endocrinology Research Center grant (DK32520) were also used.
values as per the ‘‘Relative Quantitation Method’’ outlined in
V.A. is a member of the UMass DERC (DK32520).
materials provided by Applied Biosystems (AppliedBiosystems
2008). See the Supplemental Methods for more details. Statisti-
Received June 8, 2010; accepted January 11, 2011.
cal significance was assessed by performing a t-test (two-tailed,
heteroscedastic) on the normalized Ct values of each miRNA at
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