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First published online 3 July 2008
doi: 10.1242/dev.012435
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Department of Biology, McGill University, Montréal, Québec H3A 1B1, Canada.
* Author for correspondence (e-mail: richard.roy{at}mcgill.ca)
Accepted 9 June 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: C. elegans, Notch, Dauer, Neurons, Insulin-like signalling
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
integration of signals from the surroundings sensed during the first larval
stage dictates whether the larva will progress through reproductive
development, or whether this alternative developmental pathway will be
executed. High population density initiates the dauer developmental program
through signalling by a pheromone, while high temperatures and reduced
nutrient resources strongly potentiate this decision
(Golden and Riddle, 1982;
Golden and Riddle, 1984).
However, dauer larvae can recover from this stage when growing conditions
improve, thus allowing the animal to develop into a fertile adult without
apparent morphological or reproductive consequence.
The genetic and molecular basis of dauer formation has been well
characterised and involves three highly conserved signalling pathways. These
parallel pathways (TGFβ, insulin-like and cGMP-like) affect signalling
within the nervous system of C. elegans to regulate dauer formation,
further highlighting the importance of neuronal inputs in the execution of
this developmental program (Ren et al.,
1996; Bargmann and Horvitz,
1991; Birnby et al.,
2000; Patterson and Padgett,
2000; Schackwitz et al.,
1996; Wolkow et al.,
2000). Previous studies in C. elegans have demonstrated
that different amphid neurons are important for several aspects of dauer
development, including dauer recovery, and ablation of these neurons often
fully phenocopies the abnormal dauer formation phenotypes typical of dauer
formation abnormal (Daf) mutants (Bargmann
and Horvitz, 1991; Schackwitz
et al., 1996). Recovery from the dauer stage must be equally
tightly controlled so that the post-dauer larva can resume its reproductive
developmental program and produce progeny in a suitable environment. There is,
to date, little information on how C. elegans maintains or recovers
from this stage (Tissenbaum et al.,
2000). Recovery during persistent unfavourable conditions would be
deleterious and would drastically reduce the fitness of the animal. Therefore,
the C. elegans dauer larva must constantly monitor its environment
for resource availability, pheromone level (crowding) and probably other
external signals, and must integrate all of this sensory information to elicit
the appropriate developmental response: to maintain or recover from dauer.
The Notch signalling pathway is well conserved in higher metazoans from
C. elegans to humans, and it was shown to be required in these
diverse organisms for the specification of various cell fates among a
population of equipotent cells (Bray,
2006). Recently, the Notch signalling pathway has been shown to
play a novel role in mature adult brain and in non-developmental decisions in
C. elegans, Drosophila and mouse
(Chao et al., 2005;
Costa et al., 2003;
Feng et al., 2001;
Ge et al., 2004;
Presente et al., 2004;
Yu et al., 2001). This novel
function of the Notch signalling pathway is consistent with the described
expression of some of the components of the Notch signalling pathway in
differentiated neuronal cells in adult brain
(Lee et al., 1996;
Siman and Salidas, 2004). As
these neurons are postmitotic, the requirement of the Notch signalling pathway
is unlikely to be in the specification or differentiation of neuronal
precursor cells.
We noticed that the DSL (Delta/Serrate/LAG-2) Notch ligand LAG-2 is expressed in neurons specifically at the onset of, and during, the dauer stage. As these cells are postmitotic and differentiated, the expression of the Notch ligand in head neurons reflects a possible novel role for this pathway, potentially in neuronal signalling or function. The findings we show here suggest that this initial expression of lag-2 activates canonical glp-1 Notch signalling in neurons, the function of which is crucial for the maintenance of this stage in daf-7 mutant dauer larvae. Moreover, a second Notch receptor, lin-12, is activated later in a lag-2-independent manner, and works upstream of, or in parallel with, the insulin-like signalling pathway to appropriately signal recovery from dauer and the resumption of reproductive development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The following
alleles were used: ins-18(tm339)I, rrf-3(pk1426)II,
rab-7(ok511)II/mIn1, unc-130(oy10, ev505)II, daf-7(e1372, m70,
m62)III, daf-2(e1370)III, glp-1(e2141, q224, q231)III,
lin-12d(n302, n950)III, unc-32(e189)lin-12(n676n927,
n676n930)III, lag-1(om13)IV, lag-2(q420)V and
qIs56 [lag-2::GFP, unc-119(+)]V
(Blelloch et al., 1999);
deg-1(tu38)X and kyIs51 [odr-2 2b::GFP + pJM23
(lin-15+)] (Chou et al.,
2001); and adEx1269[lin-15(+) odr-1::GFP]
(Yu et al., 1997).
lag-2 promoter variant plasmids
A 3-kb sequence upstream of the translational start of lag-2 was
cloned into pGEM-T (Promega) to generate pMR100. Different fragments were
transferred from this vector into the pPD95.67 vector using the various
enzymes indicated in Fig. 2A.
For the plasmids presented in Fig.
2B, pMR126 contains the HindIII-AccI(blunted)
fragment from pMR103 inserted into the HindIII-SmaI site of
pPD95.67. pMR133 (nucleotides -1643 to -1369 of the lag-2 promoter)
was created by PCR from pMR126 using the primer designed to delete two of the
three predicted forkhead-binding sites. pMR134 contains the PCR fragment
amplified from pMR126 (nucleotides -1520 to -1369 of the lag-2
promoter) using the primers rr289-192. pMR137 contains the PCR fragment
amplified from pMR126 using the primers rr287-288. pMR145 was created by
amplifying fragments from pMR126 with the primers rr287-294 and rr192-303.
pMR126, pMR133, pMR134, pMR137 and pMR145 were confirmed by sequencing. All
primer sequences used to create these constructs and further cloning details
are available on request.
ins-18 rescuing construct
The genomic region of the ins-18 locus, containing 
3.8 kb of
the promoter region, the coding sequence and 570 bp of the 3'UTR, was
amplified from wild-type genomic DNA with the primers rr1072-1073 using
Phusion DNA polymerase (NEB). The resulting PCR fragment was cloned into
pGEMT-t (Promega) vector (pMR1146).
UNC-130::RFP construct
The genomic region containing the unc-130 locus (promoter, ORF and
the 3'UTR) was amplified from N2 genomic DNA using the primers rr723-724
and cloned into pSKII (pMR1107). The RFP-coding region was amplified from the
plasmid dsRED2 using the primers rr615-616, digested with SacI,
blunted and cloned into the NcoI blunted site (T4 DNA polymerase) of
pMR1107 to create an in frame unc-130 C-terminal translational fusion
(pMR1107.2).
glp-1p::GLP-1::YFP reporter
The YFP variant (Nagai et al.,
2002) was amplified from the plasmid pBS7 with the primers
NheIGFP-5 and GFPSMT-3 and cloned into the HpaI-cut pGLP-1 S642N
vector (Berry et al., 1997). We
used the glp-1(gf) allele to increase expression through the
positive-feedback loop that occurs following receptor activation
(Greenwald, 1998).
Dye-filling assay
Dye filling was performed as previously described
(Burket et al., 2006). As none
of the neurons are exposed to the external environment during the dauer stage,
the larvae were stained at the L1 stage, allowed to form dauer and then imaged
thereafter.
Neuronal-specific expression of Notch receptor constructs
To create the neuronal promoter constructs, we first cloned the
glp-1 wild-type genomic sequence into pSKII and inserted a
KpnI site at the 5' end to create pMR1137.4. The neuronal
promoters (ser-2prom2, lim-6int3, gpa-2, unc-25, ceh-6, odr-1) were
amplified from wild-type genomic sequence and primers were designed to
introduce either an EagI or a NotI site at the 5' end,
in addition to an in frame KpnI site at the 3' end for cloning
into an EagI-KpnI digested pMR1137.4 vector
(Burglin and Ruvkun, 2001;
Hobert et al., 1999;
Jin et al., 1999;
Tsalik et al., 2003;
Yu et al., 1997;
Zwaal et al., 1997). All
primer sequences used to create these constructs and further cloning details
can be obtained by request.
Microinjection and transformation
All constructs were injected at concentrations ranging from 5-40 ng/µl,
with either the dominant co-injection marker pRF4 rol-6(D) or the
plasmid pMR352 (Li et al.,
2003), which expresses pharyngeal GFP under the control of the
myo-2 promoter, at 30-50 ng/µl in the corresponding genetic
background.
lag-2::GFP expression in unc-130 mutants during reproductive development
L1 larvae of the daf-2(e1370); qIs56 and unc-130(oy10);
daf-2(e1370); qIs56 were incubated at 20°C until they reached the L3
stage, after which the animals were examined for GFP expression in the IL2
neurons. The total number represents the average of three independent trials
and the bars represent the standard deviation.
Dauer assays
For the dauer recovery assay presented in
Table 1, embryos were collected
from alkaline/hypochlorite-treated gravid adults and hatched at 15°C
overnight. The resulting synchronised L1 larvae were distributed onto seeded
plates and placed at 25°C for 48 hours. For each genotype, dauers were
transferred on to pre-equilibrated plates and placed at 25°C immediately
thereafter. L4 larvae and adults were subsequently scored after 24 hours at
25°C. Each experiment was repeated at least three times.
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To assess the role of lin-12 in wild-type dauer recovery, dauers were induced by crowding/starvation and 50 animals were transferred onto plates with a fresh bacterial lawn. For the 4-hour time point, we monitored pharyngeal pumping which is an early marker for commitment to recovery. For later time points, morphological changes typical of recovering dauer larvae were scored. In both cases, the experiments were repeated five times.
For the suppression of the dauer maintenance defect by the neuronal-specific expression of glp-1, we subjected transformed gravid adults to alkaline/hypochlorite and allowed them to hatch at 15°C. The L1 larvae were then incubated at 25°C on seeded plates for two days to allow dauer formation. Then, because the transgenes were maintained as extrachromosomal arrays, we transferred 25 transformed and 25 non-transformed dauer progeny onto the same plate. We counted how many dauer larvae recovered 24 hours later for each genotype and the degree of suppression is represented as follows:
1-(recovered transformed dauer/recovered non-transformed dauer)x100.
Each experiment was performed with at least two independent lines and was repeated three times.
Laser ablation
L1 larvae were incubated at 25°C for 24 hours and laser microsurgery
was performed on early L2d animals. After the surgery, the ablated animals
were incubated for an additional 24 hours at 25°C to allow dauer formation
and the ability to maintain dauer was assayed as described above.
Microscopy and image processing
Images were captured using either a Leica DM microscope or a Zeiss LSM Meta
confocal microscope, and were processed and assembled in Photoshop CS
(Adobe).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). We observed that six head neurons express the gene
encoding the Notch DSL ligand lag-2 specifically at the onset of and
throughout the dauer stage by using a lag-2::GFP transgene
(Fig. 1A, data not shown). The
same level of lag-2::GFP expression was observed in dauer larvae
induced by dauer pheromone, starvation, or by Daf-c mutations in the three
known parallel pathways, suggesting that lag-2 expression is
activated downstream of the three major signalling pathways involved in dauer
formation (Fig. 1B). Moreover,
we did not detect any change in the expression level of the
lag-2::GFP transgene between newly induced (4 hours post-induction),
and older (96 hours post-induction) dauers, suggesting that the expression of
the Notch ligand is sustained throughout the dauer stage (data not shown).
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). The lag-2::GFP-expressing cells and the
DiI-stained neurons overlapped, leading us to conclude that lag-2 is
expressed in IL2 neurons during the dauer stage
(Fig. 1E). Consistent with
lag-2::GFP being expressed in IL2 neurons, we found that its
expression was unaffected in dauer larvae that harbour a deg-1(u38)
mutation, which causes the degeneration of the IL1 neurons early in
post-embryonic development without affecting the IL2 neurons (data not shown)
(Chalfie and Wolinsky, 1990).
Therefore, based on these results, we conclude that lag-2 is
expressed in the IL2 neurons at the onset of and throughout the dauer stage,
whether they are formed through the normal pheromone-sensing pathway due to
crowding, or by Daf-c mutations.
lag-2 expression depends on forkhead-binding sites
The expression of lag-2::GFP in IL2 neurons at the onset and
during dauer prompted us to identify the upstream components required for the
dauer-specific neuronal expression of lag-2. Deletion analysis of the
lag-2 promoter during dauer indicated that a small fragment located
1.3 kb upstream of the lag-2 translational start site was sufficient
for dauer-specific expression in IL2 neurons
(Fig. 2A). This region contains
three highly conserved forkhead transcription factor-binding sites; two of
which are nearly identical and match the FoxC consensus
(Saleem et al., 2004). We
refer to these different classes of binding sites as A and B
(Fig. 2B). To determine the
importance of these sites for the expression of lag-2, we
systematically removed each forkhead-binding site and found that they are
required for dauer/IL2-specific GFP expression
(Fig. 2C). Detailed analysis of
this region indicated that a single A-type forkhead-binding site is sufficient
for IL2-specific expression during dauer and this site is present twice in
this small interval (Fig. 2C).
Consistent with the potential role of this binding site in regulating the
appropriate lag-2 expression in these neurons, we identified a
similar `A' site within the proximal region of the promoter, which was also
sufficient to confer dauer/IL2-specific expression of lag-2
(Fig. 2A). Therefore, we
propose that, in response to environmental signals, a forkhead transcription
factor must act through these sites to trigger lag-2 expression in
the IL2 neurons at the onset of dauer.
UNC-130 is required to repress lag-2 expression during reproductive development
The genome of C. elegans is predicted to encode 15 forkhead
transcription factors, six of which have been genetically characterised
(Hope et al., 2003;
Mango et al., 1994;
Miller et al., 1993;
Nash et al., 2000;
Ogg et al., 1997) and only one
of them, the C. elegans FoxO homologue DAF-16, has been previously
shown to play a crucial role in dauer formation. However, we confirmed that
this transcription factor was not responsible for the dauer-specific
regulation of lag-2 in the IL2 neurons. First, the identified
forkhead-binding sites in the lag-2 promoter do not match the
predicted consensus DAF-16-binding site
(Furuyama et al., 2000), and,
more importantly, the dauer-dependent/IL2-specific lag-2::GFP
expression was unaffected in dauers that completely lack DAF-16
(daf-16(mgDf50) null mutant) (data not shown).
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; Mango et al.,
1994) (Fig. 3A).
Although many forkhead reporters are expressed in neurons throughout the head
region during the dauer stage, only the unc-130::GFP strain showed
strong expression in the presumptive IL2 neurons. We generated an UNC-130::RFP
translational fusion reporter construct and showed that its expression
overlapped with that of the lag-2::GFP in the IL2 neurons during
dauer (Fig. 3B). This suggests
that UNC-130 could regulate the expression of lag-2 in the IL2
neurons specifically during the dauer stage. However, we did not detect any
change in lag-2::GFP expression in unc-130 mutants during
the dauer stage (data not shown). Therefore, the UNC-130 forkhead
transcription factor is not absolutely required for dauer-specific expression
of lag-2, and/or it may function redundantly with another factor to
initiate the expression of the Notch ligand.
As unc-130::RFP is expressed in the same neurons as
lag-2, UNC-130 could be required to repress lag-2 expression
during reproductive development, which would be akin to its described
repressor role in sensory neurons
(Sarafi-Reinach and Sengupta,
2000). We therefore determined lag-2::GFP expression in
daf-2(e1370) and unc-130(oy10); daf-2(e1370), maintained at
the sub-threshold temperature for dauer formation (20°C). We noticed that,
under these sensitised conditions, 48.3±4.5% of unc-130(oy10);
daf-2(e1370) larvae misexpressed GFP in IL2 neurons during reproductive
development (n=200), compared with only 7.1±2.3% in
daf-2(e1370) mutants (n=150;
Fig. 3C). Although some Unc
mutations have been reported to affect dauer formation
(Ailion and Thomas, 2000;
Ailion and Thomas, 2003), our
observations indicate that there is no effect of unc-130 in enhancing
dauer formation (data not shown). Therefore, we suggest that the UNC-130
forkhead transcription factor is required to repress lag-2 expression
during reproductive development. Then, upon dauer formation, at least in a
daf-2 mutant background, UNC-130-mediated repression of
lag-2 is released and another transcription factor, which may bind to
the same region, is required to activate lag-2 expression.
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), we
examined the effects of mutations in Notch signalling components on
maintenance and recovery in a daf-7 mutant background, wherein the
signalling system that responds to recovery cues is competent, allowing us to
assess how Notch signals impinge on this network and affect this developmental
decision.
Under these conditions (see Materials and methods), most
(81.3±6.43%, n=416) of the daf-7(e1372);
lag-2(q420lf) dauer larvae recovered from this stage prematurely, within
24 hours following dauer formation; approximately 10-fold greater than the
baseline recovery observed in daf-7(e1372) animals
(Table 1). Moreover, laser
ablation of the IL2 neurons (which express the Notch ligand lag-2)
prior to dauer entry in a daf-7(e1372) mutant background also leads
to dauer maintenance defects that are comparable to those observed in a
daf-7; lag-2 double mutant background
(Table 2). This premature
recovery is not due to an inability of these larvae to sense pheromone, as it
can be suppressed by maintaining lag-2(q420) mutant dauers on dauer
pheromone, indicating that this premature recovery can occur only if pheromone
levels are low (Ogg et al.,
1997) (data not shown). Taken together, these findings indicate
that the DSL ligand lag-2 is expressed in the IL2 neurons at the
onset of dauer to appropriately maintain this developmental stage, and that
these neurons play an important role in dauer maintenance in
daf-7/TGFβ Daf-c mutants.
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Surprisingly, lin-12 (lf) alleles had opposite effects to
glp-1 mutants, where both lin-12(n696n927lf) and
lin-12(n696n930lf) alleles completely suppress dauer recovery in a
daf-7(e1372) mutant background
(Table 1). Furthermore, two
different lin-12 gain-of-function (gf) alleles,
n302 and n950, caused premature dauer recovery, where the
recovery frequency reflected the strength of the individual alleles
(Table 1)
(Greenwald et al., 1983).
Therefore, our data suggest that glp-1 and lin-12 signalling
play distinct roles during this developmental stage in dauers induced by a
daf-7 mutation; GLP-1 enhances dauer maintenance, whereas LIN-12
promotes timely recovery from dauer (see below and Discussion).
The downstream transcription factor LAG-1 is required for dauer recovery
LAG-1 is the C. elegans homologue of Drosophila
Suppressor of Hairless [Su(H)], and it plays an important role in directing
the Notch intracellular domain (NICD) to Notch-responsive target
genes (Christensen et al.,
1996; Jarriault and Greenwald,
2002). When we scored dauer maintenance in a
lag-1(om13lf) background, we found that only 0.6% of the
daf-7(e1372); lag-1(om13) dauer larvae recovered prematurely at the
restrictive temperature; 10-fold less than the background recovery we observed
for daf-7(e1372) mutants alone
(Table 1). Furthermore,
lag-1(om13) fully suppresses the dauer maintenance defect associated
with a glp-1(e2141) mutation, as none of the glp-1(e2141)
daf-7(e1372); lag-1(om13) dauer larvae recovered prematurely at the
restrictive temperature (Table
1). As lag-1 is downstream of both Notch receptors, its
compromise causes a consequent decrease in the expression of Notch gene
targets involved in recovery, thus rendering the animal incapable of
recovering from dauer, consistent with the phenotype observed in lin-12
(lf) mutants.
GLP-1 Notch receptor is expressed in postmitotic neurons
The nervous system is known to play a crucial role during dauer
development, so we predicted that the Notch-responding cells involved in dauer
maintenance would most likely be neurons
(Bargmann and Horvitz, 1991).
Determination of GLP-1 protein expression during the dauer stage using
antibody staining proved to be difficult because of the relatively impermeable
specialised dauer cuticle. Therefore, to detect GLP-1 in dauer larvae, we
constructed a glp-1p::GLP-1::YFP translational fusion reporter
transgene. By using this strategy, we were able to capture GLP-1::YFP
expression in head neurons located near the terminal bulb during the dauer
stage (Fig. 4A, white arrow).
The axons of these neurons project anteriorly toward the nerve ring placing
them in close proximity to the IL2 processes
(Fig. 4A, white arrowheads). In
parallel, we monitored the expression of this transgene in a
rab-7(ok511) mutant background that disrupts endosome fusion, a
process necessary for Notch turnover
(Sakata et al., 2004). In
these mutant animals, the GLP-1::YFP receptor would be internalised, but not
degraded, and therefore YFP-containing vesicles should accumulate in the
cytoplasm in a time-dependent manner, thereby facilitating our identification
of these neurons. As predicted, the GLP-1::YFP-expressing head neurons
described above accumulate YFP during the dauer stage in two bilateral head
neurons (Fig. 4B, white
arrows). GLP-1::YFP also accumulated throughout the length of the animal in
the ventral cord, in axons that appear to emanate from neurons located in the
head region (Fig. 4C, white
arrowhead). Therefore, both our genetic data, and our description of this
novel GLP-1 expression pattern in neurons, suggest that GLP-1/Notch signalling
is required in neurons in order to maintain developmental quiescence during
the dauer stage in daf-7/TGFβ mutants.
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; Hobert et al.,
1999; Jin et al.,
1999; Tsalik et al.,
2003; Yu et al.,
1997; Zwaal et al.,
1997), and we determined their capacity to rescue the dauer
maintenance defect typical of glp-1(e2141) daf-7(e1372)
(Table 3). We found that the
expression of glp-1 specifically in gpa-2- and
odr-1-expressing neurons efficiently and reproducibly suppressed the
dauer maintenance defect associated with glp-1(e2141)
(Table 3). Based on the known
expression of gpa-2 and odr-1, and on the position and
morphology of the glp-1::YFP expressing neurons, we conclude that
glp-1 is required in the AWC neurons for proper dauer maintenance.
These neurons are crucial for dauer recovery, as ablation of the AWC neurons
in a glp-1(e2141) daf-7(e1372) mutant background suppressed their
premature recovery/dauer maintenance defect
(Table 2). Moreover, although
these neurons are remodelled during the dauer stage
(Albert and Riddle, 1983), no
morphological defects were detected in the AWC neurons of glp-1(e2141)
daf-7(e1372) when compared with those of daf-7(e1372) mutants
(data not shown), suggesting that glp-1 is not required for the
dauer-specific remodelling of these neurons. However, as we observed
glp-1::YFP in the ventral nerve cord, which does not receive
processes from the AWC neurons, it is possible that glp-1 may be
required in additional neurons (Fig.
4C).
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Using pheromone, we induced animals to form dauers and then allowed them to recover by transferring these pheromone-induced or `wild-type' dauers onto plates that contained a lower concentration of dauer pheromone that permitted 70% of the wild-type dauers to recover. Under these conditions, 99.6±0.9% of the glp-1(e2141) recovered from dauer, as compared with 72.7±7.9% for wild type (Fig. 5A; P<0.005 using a Mann-Whitney test, five independent trials, n=50/trial). We did not observe a significant difference in recovery for the weaker glp-1(q231) allele in these wild-type dauers, consistent with its attenuated effect in the daf-7(e1372) background (Table 1). In addition, no significant change in the frequency of premature dauer recovery was detected using either lin-12 (lf) or (gf) mutations in this assay (Fig. 5A). It is likely that under these conditions, even in the presence of reduced pheromone concentrations, compromise of the lin-12 Notch signalling pathway cannot overcome the dauer recovery program that is presumably triggered by the resumption of insulin-like signalling. Therefore, from these data we can conclude that even in `wild-type' dauer larvae, glp-1 functions during the dauer stage to properly maintain this developmental state.
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The Notch and insulin-like signals function antagonistically during dauer
To test whether GLP-1/Notch was also required for dauer maintenance in
dauers induced through reduced insulin-like signalling
(Wolkow et al., 2000), we used
a daf-2(e1370) mutant that carries a mutation in the insulin-like
receptor. We monitored dauer recovery in both daf-2(e1370);
lag-2(q420) and daf-2(e1370) lin-12(n302gf) double mutant
backgrounds, two Notch mutations that cause premature dauer recovery in
daf-7 mutants. Unlike our findings with the daf-7/TGFβ
mutant background, none of the Notch components tested induced premature
recovery in a daf-2(e1370) mutant background (n=600 and
n=336, respectively). This suggests that the insulin-like pathway is
epistatic to both lag-2 and lin-12 during dauer development,
and that wild-type insulin-like signalling is required for the observed
premature dauer recovery associated with the lag-2 (lf) and
lin-12 (gf) mutations.
Because the DAF-2 insulin-like receptor is required for dauer recovery,
components upstream of daf-2 would be likely candidates for a signal
involved in blocking premature recovery and thus maintaining dauer.
Intriguingly, in C. elegans, there is an unusually large family of
insulin-like ligands (Pierce et al.,
2001). Two of these putative ligands, ins-1 and
ins-18, were shown to act antagonistically to the insulin-like
pathway and cause an increase in dauer formation when overexpressed
(Pierce et al., 2001).
Consistent with ins-18 acting as a target of the Notch signalling
pathway, we identified five highly conserved, putative lag-1-binding
sites within its promoter and the first intron (data not shown)
(Christensen et al., 1996).
ins-18 may therefore be activated in neurons during the dauer stage
by the Notch signalling pathway. Subsequently, it could act as a
neuroendocrine signal to prevent dauer recovery by inhibiting the insulin-like
receptor DAF-2 throughout the animal. Consistent with this possibility, we
found that the double mutant daf-7(e1372); ins-18(tm338) shows dauer
maintenance defects quite similar to Notch signalling mutants, albeit at a
lower frequency (Table 1), and
these defects can be suppressed by expressing a wild-type genomic copy of
ins-18 (Table 1).
However, we did not observe a difference in ins-18::GFP expression
during dauer stage between daf-2 and daf-2; lag-1 mutants
(data not shown). Therefore, we cannot conclude whether or not the
antagonistic insulin-like ligand ins-18 is a direct target of Notch
signalling, although our results suggest that expression of ins-18
during dauer is required, at least in part, to properly maintain dauer.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Siman and Salidas,
2004). In C. elegans, the Notch receptor gene
lin-12 is required in the adult nervous system for specific
behaviours (Chao et al.,
2005). We have demonstrated that glp-1 is required in
postmitotic neurons during dauer development in order to signal throughout the
entire animal to maintain this developmental state. When conditions improve,
lin-12/Notch signalling is required for efficient, timely recovery
from this stage. This requirement for the Notch signalling pathway during
dauer development is corroborated by microarray analyses indicating that Notch
signalling components are significantly upregulated in dauer compared with the
corresponding L3 stage (Liu et al.,
2004; Wang and Kim,
2003).
Notch functions in two distinct processes during dauer development
Our analysis of Notch function in dauer larvae induced either by a Daf-c
mutation in the daf-7/TGFβ ligand, or by pheromone/starvation,
indicates that dauer development can be divided into three distinct phases:
dauer formation, maintenance and recovery
(Fig. 6). Our model predicts
that upon dauer formation, UNC-130-dependent lag-2 repression is
alleviated, and an as yet unknown transcription factor will activate
lag-2 expression specifically in the IL2 neurons. The function(s) of
these neurons is not fully understood, but because they access the surrounding
environment they are proposed to act in chemosensation
(Riddle and Albert, 1997).
Then, throughout these adverse conditions, LAG-2 will activate GLP-1/Notch in
the AWC neurons in order to maintain this developmental stage. The AWC neurons
have been previously implicated in dauer maintenance and recovery
(Coburn et al., 1998;
Lans et al., 2004;
Zwaal et al., 1997), and
hyperactivation of gpa-2 in these neurons causes a dauer-constitutive
phenotype downstream of the cGMP signalling pathway
(Zwaal et al., 1997). The
ablation of AWC neurons blocks glp-1-dependent premature dauer
recovery, suggesting that a signal emanating from these neurons must be
released for the dauer larva to recover, and that this may be regulated by
glp-1.
Based on our observations in both starvation/pheromone-induced dauers, and
in daf-7 Daf-c dauers, we propose that once environmental conditions
improve, a second Notch ligand will activate LIN-12/Notch to promote recovery
from this stage. The cells that express both the ligand and the LIN-12/Notch
receptor remain unknown, but they may indeed be neurons. This role of
LIN-12/Notch in dauer development is consistent with previous findings
demonstrating that both sel-12 and lin-12 (lf)
mutations enhanced dauer formation in a daf-7/TGFβ mutant
background at sub-threshold temperatures
(Levitan and Greenwald, 1998).
It is quite plausible that at sub-threshold temperature (20°C),
daf-7/TGFβ mutants form transient dauers. However, if components
of the lin-12 signalling pathway were compromised, these transient
dauers would be unable to recover.
Finally, the results we obtained with the lag-1 mutation are consistent with our model, as lag-1(lf) completely suppresses the premature dauer recovery caused by reduced glp-1 function in daf-7 mutants. Because the lag-1 transcription factor acts downstream of both Notch receptors, we propose that although glp-1 downstream targets are downregulated, lin-12 targets that are important for dauer recovery are also compromised, thus preventing the dauer larva from recovering prematurely from this stage. Therefore, we have identified two distinct roles of Notch signalling in dauer development: (1) the GLP-1/Notch receptor is required to maintain dauer, probably through blocking dauer recovery in the AWC neurons; and (2) subsequent signalling through LIN-12/Notch promotes recovery.
Like the situation with lag-2, lin-12(gf) mutations do not cause
premature dauer recovery when insulin-like signalling is reduced, suggesting
that the insulin-like signalling pathway is epistatic to Notch in this
process. As the insulin-like signalling pathway is absolutely required for
dauer recovery, some of the Notch targets may include these agonistic and
antagonistic insulin-like ligands. Indeed, we have shown that mutations in the
antagonistic ins-18 insulin-like ligand cause dauer maintenance
defects similar to those of glp-1 mutants. A quick survey identified
other insulin-like ligands that possess canonical lag-1-binding
sites, which could also be regulated by Notch (data not shown)
(Christensen et al., 1996;
Rebeiz et al., 2002). Except
for ins-18, the function of these insulin-like ligands has not been
extensively studied, although some are expressed in amphid neurons, which play
an important role in dauer development
(Bargmann and Horvitz, 1991;
Pierce et al., 2001). It will
be of considerable interest to understand how these two Notch receptors are
differentially, and sequentially, activated during dauer development.
Even if insulin-like ligands are very promising targets, they may not be the only effectors involved in this novel function of the Notch signalling pathway. These additional targets would probably be different from the Notch-responding genes that become activated during cell fate specification. The identification of these downstream target genes will shed light on the role of Notch signalling in neurons during this specialised larval stage and could potentially provide a basis for the analysis of Notch-mediated processes crucial for neuronal function in higher organisms.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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