Distinct
neurogenetic
mechanisms
establish
the
same
chemosensory
valence
state
at different
life
stages
in
Caenorhabditis
elegans
Navonil
Banerjee
,
1,2
Elisa J. Rojas
Palato,
1
Pei-Yin
Shih,
3,4,5
Paul W. Sternberg
,
3
Elissa
A. Hallem
1,2,
*
1
Department
of Microbiology,
Immunology,
and Molecular
Genetics,
University
of California,
Los Angeles,
CA 90095,
USA
2
Molecular
Biology
Institute,
University
of California,
Los Angeles,
CA 90095,
USA
3
Division
of Biology
and Biological
Engineering,
California
Institute
of Technology,
Pasadena,
CA 91125,
USA
4
Present
address:
Department
of Ecology,
Evolution
and Environmental
Biology,
Columbia
University,
NewYork,
NY 10027,
USA
5
Present
address:
Zuckerman
Mind,
Brain,
Behavior
Institute,
Columbia
University,
NewYork,
NY 10027,
USA
*Corresponding
author:
Department
of Microbiology,
Immunology,
and Molecular
Genetics,
University
of California,
Los Angeles,
237 BSRB,
615 Charles
E. Young
Dr. S., Los
Angeles,
CA 90095,
USA.
Email:
ehallem@ucla.edu
An animal's
preference
for many
chemosensory
cues remains
constant
despite
dramatic
changes
in the animal's
internal
state.
The me-
chanisms
that maintain
chemosensory
preference
across
different
physiological
contexts
remain
poorly
understood.
We previously
showed
that distinct
patterns
of neural
activity
and motor
output
are evoked
by carbon
dioxide
(CO
2
) in starved
adults
vs dauers
of
Caenorhabditis
elegans
,
despite
the two life stages
displaying
the same
preference
(attraction)
for CO
2
. However,
how the distinct
CO
2
-evoked
neural
dynamics
and motor
patterns
contribute
to CO
2
attraction
at the two life stages
remained
unclear.
Here,
using
a
CO
2
chemotaxis
assay,
we show
that different
interneurons
are employed
to drive
CO
2
attraction
at the two life stages.
We also inves-
tigate
the molecular
mechanisms
that mediate
CO
2
attraction
in dauers
vs adults.
We show
that insulin
signaling
promotes
CO
2
attrac-
tion in dauers
but not starved
adults
and that different
combinations
of neurotransmitters
and neuropeptides
are used for CO
2
attraction
at the two life stages.
Our findings
provide
new insight
into the distinct
molecular
and cellular
mechanisms
used by
C. elegans
at two
different
life stages
to generate
attractive
behavioral
responses
to CO
2
.
Keywords:
carbon
dioxide
response;
Caenorhabditis
elegans
;
chemosensory
behavior;
sensory
valence;
gas sensing;
dauer
larva
Introduction
Chemosensation
is a critical
sensory
modality
that
contributes
to
locating
food,
finding
mates
or
hosts,
and
avoiding
pathogens
and
predators
(Hildebrand
and
Shepherd
1997).
In
some
cases,
the
va-
lence
of
a chemosensory
stimulus,
i.e.
whether
the
stimulus
is at-
tractive
or
aversive,
depends
on
an
animal's
physiological
state
(Ribeiro
and
Dickson
2010;
Wasserman
et al.
2012;
Wasserman
et al.
2013;
Devineni
and
Scaplen
2021).
However,
in
other
cases,
chemosensory
valence
remains
constant
despite
dramatic
changes
in
internal
physiology
(Schmidt
and
Beauchamp
1988;
Hansson
et al.
2010;
Oleszkiewicz
et al.
2022;
Banerjee
et al.
2023).
The
molecular
and
cellular
mechanisms
that
enable
animals
to
maintain
the
same
valence
state
across
widely
varying
physio-
logical
conditions
remain
poorly
understood.
The
response
of
the
free-living
nematode
Caenorhabditis
elegans
to
carbon
dioxide
(CO
2
) offers
a powerful
model
system
for
explor-
ing
the
mechanisms
that
determine
chemosensory
valence
(Banerjee
and
Hallem
2020).
While
well-fed
adults
are
repelled
by
CO
2
, starved
adults
are
attracted
to
CO
2
(Bretscher
et al.
2008;
Hallem
and
Sternberg
2008;
Bretscher
et al.
2011;
Hallem,
Spencer,
et al.
2011;
Rengarajan
et al.
2019).
In
addition,
dauer
lar-
vae—long-lived,
nonfeeding,
stress-resistant
larvae
that
form
in
response
to
adverse
environmental
conditions
(Hu
2007)—are
at-
tracted
to
CO
2
(Hallem,
Dillman,
et al.
2011a;
Banerjee
et al.
2023).
Thus,
a comparison
of
the
molecular
and
cellular
mechanisms
that
drive
CO
2
attraction
in
dauers
vs
starved
adults
can
provide
insight
into
how
different
life
stages
establish
similar
chemosen-
sory
preferences.
We
previously
showed
that
although
both
starved
adults
and
dauers
are
attracted
to
CO
2
, the
neural
activity
dynamics
and
the
motor
outputs
evoked
by
CO
2
differ
across
the
two
life
stages
(Rengarajan
et al.
2019;
Banerjee
et al.
2023).
For
example,
the
AIY,
RIG,
and
AIB
interneurons
show
different
CO
2
-evoked
calcium
re-
sponses
in
starved
adults
vs
dauers.
AIY
displays
stochastic
exci-
tatory
CO
2
-evoked
activity
in
starved
adults
(Rengarajan
et al.
2019)
but
inhibitory
CO
2
-evoked
activity
in
dauers
(Banerjee
et al.
2023),
whereas
RIG
and
AIB
do
not
respond
to
CO
2
in
starved
adults
but
display
excitatory
CO
2
-evoked
activity
in
dauers
(Rengarajan
et al.
2019;
Banerjee
et al.
2023).
However,
how
these
distinct
CO
2
-evoked
patterns
of
neural
activity
contribute
to
CO
2
attraction
was
unclear.
In
this
study,
we
correlate
the
CO
2
-evoked
activities
of
these
in-
terneurons
with
their
roles
in
promoting
CO
2
attraction
in
starved
adults
vs
dauers.
We
show
that
individual
interneurons
make
dis-
tinct
contributions
to
CO
2
attraction
at
the
two
life
stages.
Received
on
27
September
2023;
accepted
on
17
November
2023
© The
Author(s)
2023.
Published
by
Oxford
University
Press
on
behalf
of
The
Genetics
Society
of
America.
This
is an
Open
Access
article
distributed
under
the
terms
of
the
Creative
Commons
Attribution
License
(https://creativecommons.org/licenses/by/4.0/),
which
permits
unrestricted
reuse,
distribution,
and
reproduction
in
any
medium,
provided
the
original
work
is properly
cited.
G3
,
2023,
jkad271
https://doi.org/10.1093/g3journal/jkad271
Advance
Access
Publication
Date:
23
November
2023
Investigation
Downloaded from https://academic.oup.com/g3journal/advance-article/doi/10.1093/g3journal/jkad271/7444311 by California Institute of Technology user on 21 December 2023
Whereas
AIY
promotes
CO
2
attraction
in
starved
adults
but
not
dauers,
RIG
and
AIB
promote
CO
2
attraction
in
dauers
but
not
starved
adults.
We
also
show
that
insulin
signaling,
which
pro-
motes
CO
2
-evoked
AIB
activity
in
dauers
(Banerjee
et al.
2023),
functions
in
neurons
to
selectively
drive
CO
2
attraction
in
dauers.
In
addition,
we
identify
distinct
combinatorial
codes
of
neuro-
transmitters
and
neuropeptides
that
promote
CO
2
attraction
at
the
two
life
stages.
Our
results
illuminate
the
different
neuroge-
netic
mechanisms
that
operate
in
dauers
vs
adults
to
establish
the
same
chemosensory
valence
state.
Materials
and methods
C. elegans
strains
Worms
were
cultured
on
2%
nematode
growth
media
(NGM)
plates
seeded
with
Escherichia
coli
OP50
bacteria
at
ambient
temperature
(
∼
22°C)
and
CO
2
(
∼
0.038%)
as
previously
described
(Stiernagle
2006;
Scott
2011;
Banerjee
et al.
2023).
The
temperature-sensitive
strains
CB1370,
DR1565,
JT191,
EAH404,
EAH407,
and
EAH408
were
maintained
at
15°C
but
were
moved
to
ambient
temperature
(
∼
22°C)
at
least
24
h prior
to
experiments
to
minimize
any
effects
of
temperature
shifts
on
behavior,
as
pre-
viously
described
(Banerjee
et al.
2023).
The
strains
JT709
and
BQ1
were
treated
similarly
for
direct
comparison
with
strain
DR1565
in
Fig.
2f.
A
complete
strain
list
is
provided
in
Supplementary
Table
1.
Molecular
biology
and transgenesis
The
strains
where
specific
neurons
were
genetically
ablated
or
si-
lenced
were
generated
in
previous
studies
(Kunitomo
et al.
2013;
Luo
et al.
2014;
Calhoun
et al.
2015;
Jin
et al.
2016;
Guillermin
et al.
2017).
The
inx-6
AIB OFF
strain,
where
inx-6
function
was
eliminated
specifically
in
AIB,
was
generated
in
a
previous
study
(Bhattacharya
et al.
2019).
For
tissue-specific
rescue
of
daf-2
,
the
fol-
lowing
plasmids
containing
the
daf-2
cDNA
were
obtained
from
Addgene
and
individually
injected
into
the
CB1370
daf-2(e1370)
strain
at
30
ng/μL,
along
with
P
myo-2
::
dsRed
(20
ng/μL)
as
a coinjec-
tion
marker
and
pBlueScript
(50
ng/μL):
pJH4531
(P
rgef-1
::
daf-2
)
for
pan-neuronal
rescue
(Addgene
#132366),
pJH4723
(P
ges-1
::
daf-2
)
for
intestinal
rescue
(Addgene
#178899),
and
pJH4733
(P
myo-3
::
daf-2
)
for
muscle
rescue
(Addgene
#178898)
(Hung
et al.
2014).
CO
2
chemotaxis
assays
CO
2
chemotaxis
assays
on
starved
adults
and
dauers
were
per-
formed
as
previously
described
(Rengarajan
et al.
2019;
Banerjee
et al.
2023).
For
assays
with
starved
adults,
young
adults
(
∼
1
day
old)
were
washed
in
a watch
glass
and
then
starved
on
a 9 cm
2%
NGM
plate
without
bacteria
for
3 h.
Animals
were
placed
with-
in
an
annular
ring
of
Whatman
paper
soaked
in
20
mM
copper
chloride
(CuCl
2
) solution
to
prevent
the
worms
from
migrating
off
the
plate,
since
copper
is
aversive
to
C. elegans
(Campbell
et al.
2017).
After
3 h of
starvation,
the
paper
ring
was
removed.
Animals
were
then
washed
off
the
plate
into
M9
buffer
and
then
washed
twice
in
M9
and
once
in
ddH
2
O in
a watch
glass.
For
test-
ing,
animals
were
transferred
from
the
watch
glass
onto
the
cen-
ter
of
a 9 cm
2%
NGM
plate
using
a piece
of
Whatman
paper.
For
assays
with
dauer
larvae,
dauers
were
generated
by
transferring
8–10
young
adults
onto
2%
NGM
plates
with
a lawn
of
OP50
bac-
teria
and
leaving
the
plate
at
room
temperature
for
10–14
days
un-
til
the
bacterial
lawn
was
consumed.
Dauers
were
isolated
from
other
life
stages
on
the
plate
using
a sodium
dodecyl
sulfate
(SDS)
resistance
assay
(Karp
2016)
as
previously
described
(Banerjee
et al.
2023).
The
dauers
were
then
transferred
in
water
drops
onto
2%
NGM
plates
for
assays.
CO
2
chemotaxis
assays
were
performed
as
previously
described
(Guillermin
et al.
2017;
Rengarajan
et al.
2019;
Banerjee
et al.
2023).
Animals
were
placed
onto
the
center
of
a 9 cm
2%
NGM
plate.
The
CO
2
stimulus
(the
test
concentration
of
CO
2
,
21%
O
2
, balance
N
2
) and
air
stimulus
(21%
O
2
, balance
N
2
) were
pumped
through
holes
in
opposite
sides
of
the
plate
lid
at
a rate
of
2 mL/min
(for
adults)
or
0.5
mL/min
(for
dauers)
to
generate
a
CO
2
gradient
(Supplementary
Fig.
1a)
using
a syringe
pump
(PHD
2000,
Harvard
Apparatus),
with
the
syringe
output
connected
to
the
plate
lid
via
¼-inch
flexible
PVC
tubing.
Assay
duration
was
20
min
(for
adults)
or
1 h (for
dauers).
After
the
assay,
the
number
of
animals
within
a 20-mm
diameter
circle
under
each
gas
inlet
(for
adults)
or
within
30-mm
segments
on
the
side
of
the
plate
(for
dauers)
were
counted
(Supplementary
Fig.
1b
and
c).
For
transgenic
strains
with
extrachromosomal
arrays,
only
animals
expressing
the
fluorescent
transgene
were
counted.
A chemotaxis
index
(CI)
was
then
calculated
as
CI
=
#
animalsatCO
2
−
#
animalsataircontrol
#
animalsatCO
2
+
#
animalsataircontrol
.
To
account
for
directional
bias
due
to
vibration
or
other
stimuli,
assays
were
conducted
in
pairs,
with
the
gas
gradient
in
opposite
orientations.
If the
absolute
value
of
the
difference
in
CI
between
the
2 assays
in
the
pair
was
≥
0.9,
both
assays
were
discarded
as
behavior
was
assumed
to
result
from
directional
bias.
Assays
were
also
discarded
if fewer
than
7 animals
navigated
into
the
combined
scoring
regions.
For
strain
RB2575,
which
showed
de-
creased
motility,
single
assays
within
a pair
that
had
more
than
7 animals
in
the
combined
regions
were
scored
and
included
in
the
analysis
even
if fewer
than
7 animals
moved
in
the
other
assay
in
the
pair,
provided
there
was
no
directional
bias
within
the
pair.
In
the
case
of
the
neuropeptide
genes
ins-1
,
flp-2
,
and
flp-17
,
2 inde-
pendent
alleles
of
each
gene
were
tested
in
CO
2
chemotaxis
assays.
Histamine
assays
with
dauers
Transgenic
dauers
expressing
the
histamine-gated
chloride
chan-
nel
HisCl1
(Pokala
et al.
2014)
were
isolated
using
1%
SDS
treat-
ment
as
described
above.
Dauers
were
then
incubated
in
20
mM
histamine
solution
(in
dH
2
O)
for
1 h prior
to
assays.
For
the
no-
histamine
controls,
dauers
were
incubated
in
dH
2
O without
hista-
mine
for
the
same
duration.
Dauers
were
then
transferred
onto
2%
NGM
plates
without
bacteria,
with
or
without
20
mM
hista-
mine,
and
CO
2
chemotaxis
assays
were
performed
as
described
above.
Microscopy
For
starved
adults,
1-day-old
adults
were
starved
for
3 h prior
to
im-
aging.
Dauers
for
imaging
were
isolated
by
SDS
treatment
as
de-
scribed
above.
Animals
were
anesthetized
using
10
mM
levamisole
and
placed
onto
2%
agarose
pads
on
glass
slides.
Imaging
was
performed
with
a
Zeiss
Axio
Observer
inverted
wide-field
fluorescent
microscope
equipped
with
a Colibri
7 for
LED
fluorescence
illumination,
a Plan-APOCHROMAT
20
×
objective
lens,
a Hamamatsu
ORCA-Flash
4.0
camera,
and
Zen
software
(Zeiss).
For
Supplementary
Fig.
3, images
were
captured
as
z-stacks
and
maximum
intensity
projections
were
constructed
using
Fiji
(Schindelin
et al.
2012).
2 |
N.
Banerjee
et al.
Downloaded from https://academic.oup.com/g3journal/advance-article/doi/10.1093/g3journal/jkad271/7444311 by California Institute of Technology user on 21 December 2023
Statistical
analysis
Statistical
tests
were
performed
using
GraphPad
Prism
v9.3.1.
Normality
was
determined
using
the
D’Agostino–Pearson
omni-
bus
normality
test.
If data
were
not
normally
distributed,
non-
parametric
tests
were
used.
Power
analyses
were
performed
using
G*Power
v3.1.9.6
(Faul
et al.
2007).
Results
Distinct
sets of interneurons
promote
CO
2
attraction
in dauers
vs starved
adults
In
both
starved
adults
and
dauers,
CO
2
is detected
by
the
paired
BAG
sensory
neurons
in
the
head
(Hallem
and
Sternberg
2008;
Hallem,
Spencer,
et al.
2011;
Smith
et al.
2013;
Rengarajan
et al.
2019;
Banerjee
et al.
2023).
However,
many
of
the
interneurons
downstream
of
BAG
show
different
patterns
of
CO
2
-evoked
neural
activity
in
starved
adults
vs
dauers
(Fig.
1a)
(Banerjee
et al.
2023),
which
may
in
part
reflect
differences
in
synaptic
connectivity
of
these
interneurons
with
the
BAG
neurons
at
the
two
life
stages
(Fig.
1a)
(White
et al.
1986;
Varshney
et al.
2011;
Bhattacharya
et al.
2019;
Yim
et al.
2023).
To
understand
the
contribution
of
these
different
interneurons
to
CO
2
attraction
at
the
two
life
stages,
we
used
a CO
2
chemotaxis
assay
(Supplementary
Fig.
1)
to
examine
the
behavioral
responses
of
strains
where
individual
interneurons
downstream
of
BAG
were
either
genetically
ablated
or
silenced
(Carrillo
et al.
2013;
Guillermin
et al.
2017;
Katz
et al.
2018;
Rengarajan
et al.
2019).
We
focused
on
the
RIG,
AIY,
AVE,
AIB,
and
RIA
interneurons;
strains
lacking
RIB
and
AVA
function
were
not
tested
because
they
did
not
produce
enough
dauers
for
chemotaxis
assays,
indicating
a possible
role
for
these
neurons
in
promoting
dauer
entry.
We
found
that
RIG-
and
AIB-ablated
dauers
showed
reduced
CO
2
attraction
compared
with
wild
type,
whereas
AVE-silenced
dauers
were
repelled
by
instead
of
attracted
to
CO
2
(Fig.
1b,
Supplementary
Fig.
2a
and
b).
In
contrast,
the
AIY
and
RIA
neu-
rons
were
not
required
for
CO
2
attraction
in
dauers
across
all
tested
concentrations
(Fig.
1b,
Supplementary
Fig.
2c
and
d).
Thus,
RIG,
AIB,
and
AVE
promote
CO
2
attraction
in
dauers,
where-
as
AIY
and
RIA
do
not.
We
note
that
whereas
the
RIG,
AIY,
and
AIB
promoters
we
used
to
genetically
ablate
these
interneurons
showed
the
expected
cell-specific
expression
pattern
in
starved
adults
and
dauers,
the
promoter
used
to
genetically
silence
AVE
(
opt-3
)
showed
faint
expression
in
several
neurons
in
addition
to
AVE
in
dauers
(Supplementary
Fig.
3).
Thus,
we
cannot
exclude
the
possibility
that
one
or
more
of
these
opt-3
-expressing
neurons
also
contribute
to
CO
2
response
in
dauers.
To
further
investigate
the
roles
of
CO
2
microcircuit
interneur-
ons
in
regulating
CO
2
attraction
across
life
stages,
we
compared
the
behavioral
responses
of
interneuron-ablated
starved
adults
and
dauers
using
a CO
2
chemotaxis
assay.
We
found
that
wild-
type
and
AIB-ablated
starved
adults
were
similarly
attracted
to
CO
2
, suggesting
that
these
neurons
drive
CO
2
attraction
in
dauers
but
not
starved
adults
(Fig.
1c).
RIG
is also
required
for
CO
2
attrac-
tion
in
dauers
but
not
starved
adults
(Fig.
1d).
In
contrast,
AIY
is
required
for
CO
2
attraction
in
starved
adults
(Rengarajan
et al.
2019)
but
not
dauers
(Fig.
1e),
whereas
RIA
is not
required
to
estab-
lish
CO
2
attraction
at
either
life
stage
(Fig.
1b)
(Rengarajan
et al.
2019).
To
confirm
the
role
of
AIB
in
regulating
CO
2
response
in
dauers,
we
examined
the
behavioral
responses
of
dauers
in
which
the
AIB
neurons
were
chemogenetically
silenced
using
the
histamine-gated
chloride
channel
HisCl1
(Pokala
et al.
2014).
Dauers
expressing
HisCl1
specifically
in
AIB
showed
a significant
reduction
in
CO
2
attraction
when
treated
with
exogenous
hista-
mine
compared
with
untreated
control
dauers
(Fig.
1f),
confirming
that
AIB
promotes
CO
2
attraction
in
dauers.
To
further
confirm
the
role
of
RIG
in
promoting
CO
2
attraction
in
dauers,
we
hyperac-
tivated
RIG
by
expressing
a gain-of-function
allele
of
the
protein
kinase
C (
pkc-1
)
gene
(Sieburth
et al.
2005;
Sieburth
et al.
2007)
spe-
cifically
in
RIG
(Guillermin
et al.
2017).
We
found
that
hyperactiva-
tion
of
RIG
results
in
enhanced
CO
2
attraction
in
dauers
(Fig.
1g),
providing
additional
evidence
that
RIG
promotes
CO
2
attraction
in
dauers.
Thus,
both
AIB
and
RIG
promote
CO
2
attraction
in
dauers
but
not
starved
adults.
Together,
our
results
demonstrate
that
distinct
interneurons
are
required
for
establishing
the
same
valence
state
at
2 different
life
stages.
We
then
tested
the
effects
of
dauer-specific
changes
in
neuron-
al
connectivity
on
the
life
stage–specific
requirement
for
interneurons
in
establishing
CO
2
attraction.
AIB
was
previously
shown
to
form
gap
junctions
with
BAG
in
dauers
but
not
adults—the
innexin
INX-6
is
expressed
in
AIB
specifically
in
dauers,
where
it
forms
a gap
junction
complex
with
CHE-7,
a
gap
junction
subunit
expressed
in
BAG
(Fig.
2a)
(Bhattacharya
et al.
2019).
Consistent
with
previous
results
(Bhattacharya
et al.
2019),
che-7
mutant
dauers,
as
well
as
dauers
where
inx-6
expres-
sion
is eliminated
specifically
in
AIB
(
inx-6
AIB OFF
), showed
signifi-
cantly
reduced
CO
2
attraction
compared
with
wild-type
dauers
(Fig.
2b).
In
contrast
to
inx-6
AIB
OFF
dauers,
inx-6
AIB
OFF
starved
adults
showed
no
defects
in
CO
2
attraction
(Fig.
2c).
Further,
ec-
topic
expression
of
inx-6
in
AIB
did
not
affect
CO
2
responses
in
starved
adults
(Fig.
2d),
indicating
that
additional
mechanistic
dif-
ferences
underlie
CO
2
attraction
in
dauers
vs
starved
adults.
Insulin
signaling
promotes
CO
2
attraction
in
dauers
but not starved
adults
We
then
investigated
the
molecular
mechanisms
that
regulate
CO
2
attraction
in
dauers
and
starved
adults.
Insulin
signaling
reg-
ulates
entry
into
the
dauer
state
(Hu
2007)
as
well
as
a diverse
ar-
ray
of
chemosensory
behaviors
in
adults
(Tomioka
et al.
2006;
Hallem
and
Sternberg
2008;
Adachi
et al.
2010).
Moreover,
we
pre-
viously
showed
that
insulin
signaling
modulates
the
CO
2
-evoked
neuronal
activity
of
AIB
in
dauers
(Banerjee
et al.
2023).
We
there-
fore
examined
the
role
of
insulin
signaling
in
regulating
CO
2
at-
traction
in
starved
adults
and
dauers.
We
first
examined
the
CO
2
response
of
animals
carrying
a
loss-of-function
mutation
in
the
daf-2
gene,
which
encodes
the
sole
C. elegans
insulin
receptor
(Hu
2007).
We
found
that
loss
of
daf-2
func-
tion
had
no
effect
on
CO
2
attraction
in starved
adults
(Fig.
2e).
In
con-
trast,
daf-2
mutant
dauers
were
unresponsive
to
CO
2
, suggesting
that
daf-2
promotes
CO
2
attraction
in
dauers
(Fig.
2e–f).
The
daf-2
gene
is
broadly
expressed
in
multiple
tissues
in
both
adults
and
dauers
(Murphy
and
Hu
2013;
Martinez
et al.
2020).
To
identify
the
tissues
where
daf-2
might
function
to
regulate
CO
2
attraction
in
dauers,
we
performed
CO
2
chemotaxis
assays
on
daf-2
mutant
dauers
in
which
DAF-2
function
was
specifically
restored
in
either
neurons,
intestine,
or
muscle.
Restoring
DAF-2
function
in
neurons,
but
not
intestine
or
muscle,
partially
yet
significantly
rescued
the
chemotaxis
defect
of
daf-2
mutant
dauers
(Supplementary
Fig.
4).
These
results
suggest
that
DAF-2
primarily
functions
in
neurons
to
mediate
CO
2
attraction
in dauers.
In
addition,
we
found
that
dauers
carrying
loss-of-function
mutations
in
the
3-phosphoinositide-dependent-kinase-1
gene
pdk-1
and
the
protein
kinase
B (Akt/PKB)
gene
akt-1
,
both
of
which
act
downstream
of
daf-2
(Hu
2007),
showed
significantly
reduced
CO
2
at-
traction
(Fig.
2f).
Thus,
insulin
signaling
promotes
CO
2
attraction
in
dauers
but
not
starved
adults.
Neurogenetic
mechanisms
of
chemosensory
valence
| 3
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