PNAS
2024 Vol. 121 No. 40 e2405391121
https://doi.org/10.1073/pnas.2405391121
1 of 12
RESEARCH ARTICLE
|
Significance
Animals must respond
appropriately to multiple sensory
stimuli to make informed
decisions. It remains unclear how
the nervous system is able to
integrate different sensory cues
and propagate that information
toward making decisions over
longer timescales. We use the
nematode
Caenorhabditis elegans
to investigate how sensory
integration occurs during the
decision to exit diapause, a
stress
-
resistant developmentally
arrested state triggered by
multiple sensory inputs including
food availability and population
density. We show how expression
of an insulin
-
like peptide critical to
dauer exit reflects the sensory
integration process and decision
commitment, and we dissect the
regulation of this insulin
-
like
peptide’s expression. Our study
analyzes the relationship
between neuronal activity and
neuropeptide expression during
a complex decision with diverse
sensory inputs.
Author contributions: M.G.Z., M.S., A.T., V.V., and P.W.S.
designed research; M.G.Z., M.S., S.H.M., A.T., and
N.F. performed research; M.G.Z., M.S., H.P., and F.C.S.
contributed new reagents/analytic tools; M.G.Z. and M.S.
analyzed data; and M.G.Z. and P.W.S. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Copyright © 2024 the Author(s). Published by PNAS.
This article is distributed under
Creative Commons
Attribution
-
NonCommercial
-
NoDerivatives License 4.0
(CC BY
-
NC
-
ND)
.
1
To whom correspondence may be addressed. Email:
pws@caltech.edu.
This article contains supporting information online at
https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
2405391121/-
/DCSupplemental
.
Published September 24, 2024.
NEUROSCIENCE
Sensory integration of food and population density during
the diapause exit decision involves insulin
-
like signaling
in
Caenorhabditis elegans
Mark G. Zhang
a
, Maedeh Seyedolmohadesin
b
, Soraya Hawk Mercado
a
, Arnaud Tauffenberger
c
,d
, Heenam Park
a
, Nerissa Finnen
a
,
Frank C. Schroeder
c
,d
, Vivek Venkatachalam
b
, and Paul W. Sternberg
a,1
Affiliations are included on p. 10.
Edited by Gary Ruvkun, Massachusetts General Hospital, Boston, MA; received March 19, 2024; accepted August 20, 2024
Decisions made over long time scales, such as life cycle decisions, require
coordinated
interplay between sensory perception and sustained gene expression. The
Caenorhabditis
elegans
dauer (or diapause) exit developmental decision requires sensory integration of
population density and food availability to induce an all
-
or
-
nothing organismal
-
wide
response, but the mechanism by which this occurs remains unknown. Here, we
demonstrate how the Amphid Single Cilium J (ASJ) chemosensory neurons, known
to be critical for dauer exit, perform sensory integration at both the levels of gene
expression and calcium activity. In response to favorable conditions, dauers rapidly
produce and secrete the dauer exit
-
promoting insulin
-
like peptide INS
-
6. Expression
of
ins
-
6
in the ASJ neurons integrates population density and food level and can reflect
decision commitment since dauers committed to exiting have higher
ins
-
6
expression
levels than those of noncommitted dauers. Calcium imaging in dauers reveals that
the ASJ neurons are activated by food, and this activity is suppressed by pheromone,
indicating that sensory integration also occurs at the level of calcium transients.
We find that
ins
-
6
expression in the ASJ neurons depends on neuronal activity in
the ASJs, cGMP signaling, and the pheromone components ascr#8 and ascr#2. We
propose a model in which decision commitment to exit the dauer state involves an
autoregulatory feedback loop in the ASJ neurons that promotes high INS
-
6 produc-
tion and secretion. These results collectively demonstrate how insulin
-
like peptide
signaling helps animals compute long
-
term decisions by bridging sensory perception
to decision execution.
dauer | diapause | sensory | integration | insulin
A fundamental goal of neuroscience is to understand how nervous systems sense, integrate,
and interpret diverse stimuli to deliver the appropriate output. The duration over which
these processes occur spans from shorter timescales, such as during reflexive responses to
harsh stimuli or chemotaxis toward attractive stimuli ( 1 – 5 ), to longer timescales, such as
migration or hibernation decisions in response to changing weather patterns, temperature,
and food availability ( 6 – 9 ). We lack a clear understanding of how neuronal activity, which
occurs over short timescales of milliseconds to seconds, informs downstream processes
that occur over timescales of hours or longer. One such decision that takes place over a
longer timescale is the developmental decision to enter (or exit) diapause, a temporarily
suspended developmental state that protects against environmental stress and promotes
dispersal. Diapause is evolutionarily conserved across metazoans and requires integration
of environmental cues to inform a coordinated, organismal
-
wide decision ( 10 – 12 )
To better understand how a compact nervous system can interpret sensory signals to
coordinate an organismal
-
wide decision that takes place over multiple hours, we studied
the dauer exit decision. During early larval growth,
C. elegans
choose between two devel-
opmental fates depending on environmental conditions ( Fig.
1
A
). Under favorable con-
ditions, larvae undergo reproductive growth, whereas under unfavorable conditions, larvae
enter the stress
-
resistant, long
-
lived developmentally arrested diapause state known as
dauer ( 13 – 15 ). While in the dauer state,
C. elegans
continually assess their surroundings
to detect environmental improvement; when conditions sufficiently improve, animals exit
the dauer state and return to the reproductive cycle as late
-
stage larvae. Of the various
environmental inputs to the dauer entry and exit decisions, the strongest input is a ratio
of food to pheromone ( 14 ). Here, “pheromone” refers to a mixture of secreted dauer
-
regulating signaling molecules, termed ascarosides, that collectively convey population
density ( 16 , 17 ).
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The genetic pathways and neuroendocrine signaling mechanisms
that govern the dauer entry and exit decisions ( 18 – 20 ) including
a cGMP pathway, a TGF
- β-
like pathway, an insulin/insulin
-
like
growth factor (IGF)
-
1 signaling (IIS) pathway, and a steroid hor-
mone pathway. While considerably more attention has been paid
toward the dauer entry decision, previous studies identified two
insulin
-
like peptides, INS
-
6 and DAF
-
28, as regulators of dauer
exit that work within the IIS pathway ( 21 , 22 ).
Despite the wealth of knowledge collected on the
C. elegans
dauer entry and exit decisions, many fundamental questions
remain, including: 1) How does sensory integration of food, pher
-
omone, and temperature (among other possible cues) occur? and
2) How does short
-
term perception of environmental cues trans-
late into the dauer decisions which take place over the course of
hours? To address these questions, we studied the dauer exit deci-
sion using genetic and molecular neurobiological approaches.
Using an ethologically relevant, pheromone
-
based assay, we
demonstrate an essential role for neuropeptide signaling as a whole
in dauer exit and validate INS
-
6 as important for dauer exit.
To understand how INS
-
6 production relates to sensory per-
ception, we analyzed the spatiotemporal dynamics of
ins
-
6
expres
-
sion in response to different environmental cues and found that
ins
-
6
expression in a pair of chemosensory neurons, the Amphid
Single Cilium J (ASJ) neurons, reflects both food and pheromone
levels during dauer exit. We found that high
ins
-
6
expression in
the ASJ neurons reflects commitment to exit the dauer state.
Through calcium imaging in dauers, we show that sensory inte-
gration of food and pheromone can also be seen at the level of
sensory neuron activity: ASJ calcium levels increase in response
to food, but this response can be suppressed by adding phero-
mone. We find that
ins
-
6
upregulation during dauer exit depends
on ASJ neuronal activity, cGMP signaling, a CaM
-
kinase pathway,
and is inhibited most potently by the pheromone component
ascr#8. Altogether, our data show how the ASJ neurons integrate
food and pheromone levels both in the short
-
term through cal-
cium transients and in the longer term through transcription of
ins
-
6
, thereby highlighting how neuropeptides can provide the
bridge from ephemeral sensory information to sustained physio-
logical changes.
Results
A Screen of ASJ
-
Enriched Neuropeptide Genes Validates
ins
-
6
as Critical for Dauer Exit.
Neuronal ablation methods using
a laser microbeam or transgenic caspases have shown that the
chemosensory ASJ neurons are important for dauer exit (21,
23, 24), but the precise molecular mechanism by which the ASJ
neurons promote dauer exit remains unclear. Having previously
shown that neuropeptides are collectively required for dauer entry
(25), we reasoned that neuropeptides could also be involved in
dauer exit. Using a pheromone
-
based dauer exit assay (Fig. 1
B
), we
tested five mutants defective in neuropeptide synthesis or secretion
and found that four out of the five loss
-
of
-
function mutants (
sbt
-
1/7BT
,
egl
-
3/PC2
,
egl
-
21/CPE
, and
unc
-
31/CAPS
) had significantly
lower dauer exit rates than that of wild
-
type (Fig. 1
C
), strongly
indicating that neuropeptide signaling is required for exit. By
contrast,
ric
-
7
loss
-
of
-
function mutants, which are impaired for
dense core vesicle secretion (26), exited dauer at rates higher than
A
CD
B
Fig. 1.
Neuropeptides, especially the insulin
-
like peptide
ins
-
6
, are critical for dauer exit. (
A
) During development,
C. elegans
makes multiple developmental
decisions including whether to enter and when to exit the developmentally arrested state called dauer. This decision depends mainly on food availability, crowding,
and temperature. (
B
) Overview of the dauer exit assay. Animals were induced to become dauer via growth on conditions of high pheromone concentration and
high temperature. Nondauers were removed using SDS and surviving dauers were transferred to conditions of intermediate pheromone concentrations and
lower temperature to stimulate between 40 and 80% of dauers to exit after 24 h. (
C
) Dauer exit rates of mutants defective for neuropeptide processing (
sbt
-
1
,
egl
-
3
,
egl
-
21
) or signaling (
unc
-
31
,
ric
-
7
) compared to that of wild type (WT). Means are written and shown by the orange line, and total number of animals scored
is indicated in parentheses. (
D
) Dauer exit rates of mutants defective for single neuropeptide genes. Data compiled from multiple independent experiments
and statistical analyses were only performed between mutant and wild
-
type samples measured in the same experiment. Bars indicate means. See
SI Appendix
,
Fig. S1
for analysis of individual mutants. For (
C
) and (
D
), each dot is the dauer exit % from an assay plate containing 50 to 100 animals each. ****
P
< 0.0001,
***
P
< 0.001, **
P
< 0.01, compared to “WT” by Welch ANOVA with Dunnett’s T3 multiple comparison correction. Comparisons to “WT” were not statistically
significant unless indicated otherwise.
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PNAS
2024 Vol. 121 No. 40 e2405391121
https://doi.org/10.1073/pnas.2405391121
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that of wild
-
type, suggesting that RIC
-
7 may impair the secretion
of different neuropeptides than those affected by the other four
mutants.
We hypothesized that the ASJ neurons may utilize neuropep-
tides to promote dauer exit and tested this by screening for defects
in dauer exit rates in mutants lacking ASJ
-
enriched neuropeptides.
We analyzed loss
-
of
-
function mutants for eleven insulin
-
like pep
-
tide genes, two neuropeptide
-
like peptide genes, and two
FMRF
-
amide
-
like peptide genes that were predicted to be enriched
in ASJ based on single
-
cell RNA
-
sequencing data ( 27 ) ( Fig.
1
D
and
SI Appendix
, Fig. S1
A
). Of the fifteen genes tested,
ins
-
6
mutants showed the most severe dauer exit defect as they exited
dauer at the lowest rates compared to wild type.
ins
-
32
mutants
showed a modest dauer exit defect, while mutants defective for
ins
-
26
,
nlp
-
80
,
flp
-
15
, or
flp
-
34
showed higher than wild
-
type
dauer exit rates, suggesting that the neuropeptides those genes
encode may inhibit dauer exit. Future cell
-
specific knockdown
experiments in ASJ will help to ascertain whether
ins
-
6
acts pri-
marily from ASJ vs. other neurons.
ins
-
6
Transcription and INS
-
6 Secretion Increase Quickly during
Dauer Exit.
We focused on characterizing the expression pattern of
ins
-
6
because loss of
ins
-
6
resulted in the most severe impairment
in dauer exit of the neuropeptide genes we tested. To characterize
how
ins
-
6
expression responds to environmental improvement
during dauer exit, we analyzed
ins
-
6
transcription and INS
-
6
secretion using genomically integrated fluorescence reporters
that were simultaneously integrated into the same strain (Fig. 2
A
and
SI Appendix
, Table S1
). To measure
ins
-
6
transcription, we
constructed
ins
-
6p::destabilized
-
YFP (dYFP)
, a transcriptional reporter
based off a previous design (21) that fuses both upstream and downstream
regulatory regions of
ins
-
6
to dYFP, a modified YFP variant with a shorter
half
-
life that provides higher temporal resolution (28). To measure INS
-
6
secretion, we constructed
ins
-
6::mCherry
, a translational reporter that
uses the
ins
-
6
promoter to drive expression of a fusion between the
INS
-
6 propeptide and mCherry, a protein whose secretion can be
tracked using a coelomocyte uptake assay (29) in which fluorescence
intensity within the coelomocytes reflects the amount of neuropeptide
secreted into the body cavity. We built a strain containing simultaneously
A
C
HI
J
DE
FG
B
Fig. 2.
ins
-
6
transcription and INS
-
6 secretion increase quickly during dauer exit. (
A
) Design of
ins
-
6
transcriptional and translational reporters. (
B
) Sample
images of merged dYFP and mCherry channels.
Left
, dauer prior to transfer.
Right
, dauer 6 h after transfer. Anterior is
Left
, ventral is facing the viewer. (
C
–
E
)
Quantification of
ins
-
6p::
dYFP transcriptional reporter activity measured in arbitrary units (a.u.) in ASJ (
C
) or ASI (
D
) and mCherry signal in the coelomocytes (
E
) in
dauers and following transfer of dauers to favorable conditions (indicated by curved arrow). Plots are from the same set of animals. ns, not significant. *
P
< 0.05,
**
P
< 0.01, ***
P
< 0.001, ****
P
< 0.0001 by Welch ANOVA with Dunnett’s T3 multiple comparison correction when compared to “Dauer.” (
F
) Representative images
of mRNA FISH analysis for
ins
-
6
mRNA in a dauer and an exiting dauer 3 h after transfer (h.a.t.) to favorable conditions. (
G
) mRNA FISH signal quantification in
the ASJ neurons measured in arbitrary units (a.u.). *
P
< 0.05, **
P
< 0.01 by Welch ANOVA with Dunnett’s T3 multiple comparison correction when compared
to “Dauer.” (
H
–
J
) Additional
ins
-
6
reporter activity experiments performed similarly to (
C
–
E
), except with additional time points, and LED power and exposure
time were lowered relative to the experiments from (
C
–
E
) to prevent pixel saturation. “Dauer (early)” and “Dauer” refer to dauer animals obtained 48
-
and 65
-
h
after incubation, respectively. h.a.t., hours after transfer to favorable conditions. In all plots, individual dots represent one animal. Medians are depicted by the
orange bar. Medians and sample sizes are written.
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integrated copies of both these reporters (
SI Appendix
, Table S1
) and
concurrently measured YFP signal in head neurons and mCherry
signal in coelomocytes (Fig. 2
B
).
ins
-
6
transcriptional reporter activity was nearly undetectable
in dauers ( Fig.
2
C
and
D
), although we did observe a slight
increase in reporter activity in the ASJ neurons of animals that
recently entered dauer ( Fig.
2
H
), in accordance with previous
observations ( 21 ) (
Discussion
). This signal then returned to base-
line after animals remained in the dauer state for
~
12 h. When
these dauers were transferred to favorable conditions to induce
dauer exit,
ins
-
6
transcriptional reporter activity in the ASJ neu-
rons increased after just 1 h ( Fig.
2
C
) and continued to increase
for multiple hours thereafter ( Fig.
2
C
and
F
).
ins
-
6
transcriptional
reporter activity also increased in the ASI chemosensory neurons
albeit at a slower rate and to a lower maximum relative to the ASJ
neurons ( Fig.
2
D
and
G
). To validate these results, we performed
fluorescence in
situ hybridization (FISH) for
ins
-
6
mRNA ( Fig.
2
F
and
G
). Consistent with our fluorescence reporter results, our
FISH data indicated that
ins
-
6
transcripts increased just 1 h after
transfer and continued to increase afterward. Concordant with
results using our
ins
-
6
transcriptional reporter, we observed that
ins
-
6::mCherry
translational reporter activity in the coelomocytes
increased within the first hour following transfer to favorable con
-
ditions and continued to increase for multiple hours thereafter
( Fig.
2
E
and
H
).
Under reproductive growth conditions, which result in non-
dauer adult development, our
ins
-
6
transcriptional reporter showed
no signal in ASJ throughout development (
SI Appendix
, Fig.
S2
A
).
ins
-
6
transcriptional reporter activity in ASI (
SI Appendix
, Fig.
S2
B
)
and translational reporter activity (
SI Appendix
, Fig. S2
C
) in the
coelomocytes peaked in L1 larvae and then decreased throughout
development. These results suggest that favorable conditions cause
increased
ins
-
6
reporter activity in ASJ specifically in the context
of dauer animals.
The observed increase in INS
-
6 secretion could result from an
increase in
ins
-
6
transcription, INS
-
6 processing, INS
-
6 packag-
ing, and/or dense core vesicle release. To parse these different fac-
tors, we built a translational reporter driven by a constitutively
active ASJ
-
specific promoter,
trx
-
1p::ins
-
6::mCherry
, and again
measured coelomocyte uptake of INS
-
6::mCherry (
SI Appendix
,
Fig. S2
D
). The mCherry signal in coelomocytes increased when
dauers were transferred to favorable conditions in a manner similar
to animals bearing the
ins
-
6p::ins
-
6::mCherry
reporter transgene,
suggesting that secretion of INS
-
6 is also controlled at the level
of dense core vesicle release.
daf
-
28
Expression Patterns Suggest a Weaker Role in Dauer Exit
versus
ins
-
6
.
DAF
-
28, another insulin
-
like peptide that promotes
nondauer development during the dauer entry decision (30), has
previously been shown to be a weaker regulator of dauer exit
relative to INS
-
6 (21, 22). Consistent with those observations,
loss of
daf
-
28
gene function did not result in a defect in dauer
exit rates in an otherwise wild
-
type background but did enhance
the dauer exit rate defect of
ins
-
6
loss
-
of
-
function mutants
(Fig. 1
D
and
SI Appendix
, Fig. S1
B
). To understand how the
spatiotemporal regulation of
daf
-
28
compares with that of
ins
-
6
,
we performed similar transgenic reporter experiments to study
daf
-
28
(
SI Appendix
, Fig. S2
E
–
H
).
A
daf
-
28p::dYFP
transcriptional reporter showed virtually no
activity in the ASJ neurons (
SI Appendix
, Fig.
S2
F
) of dauers after
being transferred to favorable conditions but did show slight
activity increase in the ASI neurons (
SI Appendix
, Fig. S2
G
).
daf
-
28::mCherry
translational reporter activity measured in the
coelomocytes of dauers did not increase even 6 h after transfer
to favorable conditions (
SI Appendix
, Fig. S2
H
). In contrast,
growth under nondauer
-
inducing conditions resulted in strong
daf
-
28p::dYFP
transcriptional reporter activity in the ASI neu-
rons (
SI Appendix
, Fig.
S2
G
), weaker activity in the ASJ neurons
(
SI Appendix
, Fig.
S2
F
), and strong activity in the coelomocytes
(
SI Appendix
, Fig.
S2
H
), with activity for both reporters peaking
after animals reached adulthood. Collectively, our transgenic
reporter lines for
ins
-
6
and
daf
-
28
show inverse expression pat-
terns:
ins
-
6
reporter activity increases during dauer exit but less
so during reproductive growth, and vice versa for the
daf
-
28
reporters. These findings align with both ours’ and others’ dauer
exit assay results in which INS
-
6 plays a stronger role in regulat-
ing dauer exit than does DAF
-
28 ( 21 , 22 ).
ins
-
6
Expression Reflects Commitment in the Dauer Exit
Decision.
ins
-
6
expression increases quickly in all dauers when
they are transferred to strongly favorable conditions that stimulate
100% of dauers to exit, but what happens when conditions are
more ambiguous such that only a fraction of dauers exit? We
transferred dauers to a lower pheromone concentration that
induces approximately half of dauers to exit (hereby referred
to as “intermediate
-
pheromone conditions”) and measured
ins
-
6p::dYFP
transcriptional reporter activity (Fig. 3
A
). At 3 h after
transfer, all worms showed a slight increase in
ins
-
6p::dYFP
signal,
while at 6 h after transfer and beyond, a clear bimodality of the
population emerges: The worms that would eventually go on to
exit dauer [as evidenced by a wider pharynx (Fig. 3
B
), which is a
key morphological characteristic of dauer exit (31)], showed high
ins
-
6
transcriptional dYFP reporter activity, whereas the worms
that should remain as dauers showed a return to baseline levels of
dYFP signal, resembling the level of signal seen in dauers.
To determine whether
ins
-
6
expression could be used to
distinguish dauers that are committed versus noncommitted,
we compared the time course of
ins
-
6
expression with that of
dauer exit commitment ( Fig.
3
C
). To examine dauer commit-
ment, we transferred dauers from high
-
pheromone, dauer
-
maintaining conditions to intermediate
-
pheromone conditions
that permitted approximately half the animals to exit dauer. Then,
after different time intervals, we transferred the animals back
onto high pheromone conditions so that animals that had not
committed to exiting dauer would be induced to remain as dauers
( Fig.
3
C
,
To p
); this protocol was analogous to previous work that
established commitment to exiting dauer ( 13 ). Within 3 h of
exposure to intermediate
-
pheromone conditions, approximately
half the animals that might exit dauer under such conditions
committed to exiting dauer (18% committed, compared to a
baseline of 42%), while roughly the total pool of dauers that
would eventually exit under intermediate
-
pheromone conditions
committed within 6 h (43%) ( Fig.
3
C
). These observations indi
-
cate that while some dauers irreversibly commit in 1 h following
transfer to intermediate
-
pheromone conditions, other dauers can
take between 3 and 6 h to commit to the exit decision. This 6
-
h
mark matches the time point in our imaging experiments in
which the clearest bimodality of both
ins
-
6p::dYFP
signal as well
as pharynx width emerges ( Fig.
3
B
), suggesting that such bimo-
dality can reflect decision commitment.
ins
-
6
Expression Reflects a Food:Pheromone Ratio.
Since
ins
-
6
transcriptional reporter activity responded differently when dauers
were transferred to no
-
pheromone versus intermediate
-
pheromone
conditions (compare Fig. 2
C
to Fig. 3
A
), we asked whether
ins
-
6
expression responds to a food:pheromone ratio—the principal
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