Regulation of Behavioral Plasticity by Systemic Temperature Signaling in Caenorhabditis Elegans

8/3/2019 Regulation of Behavioral Plasticity by Systemic Temperature Signaling in Caenorhabditis Elegans

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984 VOLUME 14 | NUMBER 8 | AUGUST 2011 nature neurOSCIenCe

a r t I C l e S

Temperature is an unavoidable somatic stimulus that ultimately

affects survival and reproduction. The impact of temperature hasbeen clearly demonstrated by studies showing that change of core

body temperature by means of changing the temperature of thehypothalamus alters the lifespan of transgenic mice1. To confront

environmental temperature change, animals must evolve a behavioralstrategy for ensuring the temperature homeostasis in their bodies.

This evolution is exemplified by poikilotherms, whose body tempera-ture is determined by temperature exchange with the surrounding

environment owing to the large surface area to volume ratios of their

bodies

2

. Various poikilotherms have acquired the ability to memorizecomfortable temperatures and to move to places that minimize tem-perature deviation from the memorized temperatures.

One such successful strategy of poikilotherms is observable inthermotaxis in the nematode C. elegans3,4: most worms that are cul-

tivated at a particular temperature between 15 C and 25 C for a fewhours with a food source migrate to the cultivation temperature when

placed on a thermal gradient for an hour5,6. In addition, worms thatare conditioned to migrate to a certain temperature are capable of

migrating to a new cultivation temperature a few hours after shiftingto the new cultivation temperature3,5. Previous studies provided a

neural model for thermotaxis, in which temperature is sensed, pro-cessed and remembered through a neural circuit that is composed of

thermosensory neurons and interneurons4,79.

Here we show that systemic temperature signaling induces modula-tion of the thermotactic neural circuit in C. elegans. Genome-wideprofiling upon behavioral conditioning indicated that perception of

a change in normal cultivation temperatures by heat-shock transcrip-

tion factor HSF-1, known for its responsiveness to transient heat-shock stimuli (between approximately 30 C and 35 C, where the

range of normal cultivation temperatures in C. elegans is betweenapproximately 15 C and 25 C), drives the expression dynamics of

direct or indirect downstream genes not found among the classical

heat-shock response hsp genes. Expressions of HSF-1 in body wallmuscles or intestine restored the thermotactic defect ofhsf-1 mutants,

suggesting that non-neuronal cells can induce HSF-1-dependentdownstream signaling in accordance with cultivation temperature

changes. Genetic epistasis analysis and calcium imaging showed thatthis systemic signaling regulates non-cell autonomously the thermo-

sensory neurons through the estrogen signaling pathway, leading tobehavioral change.

RESULTS

Genome-wide profiling highlights HSF-1

To gain detailed molecular insight into thermotactic behavior, we

performed a genome-wide microarray analysis during behavioralconditioning and compared the transcriptional profile of worms con-

ditioned to migrate to the new temperature of 17 C with worms con-ditioned to migrate to the previous temperature of 23 C (Fi. 1a andSuppary Fis. 1 and 2). We detected 79 candidate genes, whichare likely to include genes that are involved in thermotactic behavior

in addition to genes that are differentially expressed simply as a resultof temperature shift. Examining the candidate genes, we noticed sub-

stantial changes in expression of several hsp genes, which are regulatedby HSF-1 (ref. 10). HSF-1 is highly conserved from nematodes to mam-

mals11. Previous structural and functional characterizations of HSF-1

revealed that the transient heat-shock stimuli enable HSF-1 to changestructurally from an autoinhibited monomer to a trimer and to trans-locate to the nucleus, thereby activating gene transcription10,1216. Thus,

HSF-1 is well known to directly perceive transient heat-shock stimuli.

Notably, previous in vitro studies have shown that normal growth tem-peratures, as well as transient heat-shock stimuli, can induce the reversi-

ble structural transition of HSF-1 and its DNA binding17,18. Consideringthese previous observations and our own (Suppary Fi. 2),

1Group o Molecular Neurobiology, Graduate School o Science, Nagoya University, Nagoya, Japan. 2CREST, Japan Science and Technology Agency, Tokyo, Japan.3Institute or Advanced Research, Nagoya University, Nagoya, Japan. 4Present address: Department o Molecular Engineering, Graduate School o Engineering, Kyoto

University, Kyoto, Japan. Correspondence should be addressed to I.M. ([email protected]).

Received 1 April; accepted 3 May; published online 26 June 2011; doi:10.1038/nn.2854

Regulation of behavioral plasticity by systemictemperature signaling in Caenorhabditis elegans

Takuma Sugi1,4, Yukuo Nishida1 & Ikue Mori13

Animals cope with environmental changes by altering behavioral strategy. Environmental information is generally received by

sensory neurons in the neural circuit that generates behavior. However, although environmental temperature inevitably influences

an animals entire body, the mechanism of systemic temperature perception remains largely unknown. We show here that

systemic temperature signaling induces a change in a memory-based behavior in C. elegans. During behavioral conditioning,

non-neuronal cells as well as neuronal cells respond to cultivation temperature through a heat-shock transcription factor that

drives newly identified gene expression dynamics. This systemic temperature signaling regulates thermosensory neurons

non-cell-autonomously through the estrogen signaling pathway, producing thermotactic behavior. We provide a link betweensystemic environmental recognition and behavioral plasticity in the nervous system.
http://www.nature.com/doifinder/10.1038/nn.2854http://www.nature.com/doifinder/10.1038/nn.2854


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nature neurOSCIenCe VOLUME 14 | NUMBER 8 | AUGUST 2011 98 5

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we wished to address the new biological importance of the role ofHSF-1 in responding to normal cultivation temperatures.

We confirmed in vivo whether a temperature shift within a range of

ambient cultivation temperatures (between approximately 15 C and25 C) activates C. elegans HSF-1 by observing the green fluorescentprotein (GFP) fluorescence of wild-type strains expressing hsp-16.2

promoter::gfp reporter, whose promoter contains two heat-shock tran-scription factor binding elements (HSEs) (Fi. 1). We observed large

GFP fluorescence changes that were dependent on two HSEs at 2 hafter shifting the temperature from 15 C to 23 C or to 25 C (Fi. 1fh

and Suppary Fi. 3a), which were comparable with thoseobserved at 2 h after the transient heat-shock treatment (Fi. 1i).

As HSF-1 activity responded to ambient cultivation temperatures,we addressed whether HSF-1 is required for thermotactic behavior.

hsf-1(sy441) mutants19, carrying a reduction-of-function mutation(see Suppary Srais a pasis), migrated to a cultiva-

tion temperature to which they had previously been habituated when

cultivated at 17 C (Suppary Fi. 4a,b,), whereas mutantscultivated at 23 C did not migrate to the cultivation temperature(Suppary Fi. 4a,,). In a temperature shift assay, at 5 h after

shifting the cultivation temperature from 17 C to 23 C, hsf-1(sy441)

mutants failed to migrate toward higher temperatures (23 C) andinstead migrated to colder temperatures (Fi. 2a). At 5 h after shift-

ing the cultivation temperature from 23 C to 17 C, they showed atendency to migrate toward the new cultivation temperature of 17 C

(Fi. 2b). Worms carrying the hsf-1(ok600) null mutation as either ahomozygote or heterozygote showed a behavioral defect similar to

that of the hsf-1(sy441) mutants (Fi. 2).We next addressed whether the abnormal thermotactic migration

ofhsf-1(sy441) mutants is merely a reflection of slower temperature

memory acquisition: might they eventually become conditionedto the higher temperatures if cultivated at them longer? This was,

however, not the case, since hsf-1(sy441) mutants also showed the

behavioral defect at 10 h after shifting the temperature from 17 Cto 23 C (Fi. 2).Excess expression of native HSF-1 in wild-type worms10 also

induced partially abnormal migration, which was partly reminiscentof the defect ofhsf-1(sy441) mutants (Fi. 2a,b). However, hsf-1(sy441)

mutants grown at 23 C showed cryophilic behavior of greater sever-ity than hsf-1 overexpression lines grown at 23 C, which probably

leads to the behavioral difference observed after shifting the cultiva-tion temperature to 17 C. When cultivated for a longer time after

shifting temperature from 17 C to 23 C, hsf-1(sy441) mutants alsoshowed a strong tendency to migrate toward colder regions than hsf-1

overexpression lines, although that difference was not statisticallysignificant (Fi. 2). These observations suggest that hsf-1(sy441)

mutants and hsf-1 overexpression lines both show an abnormal cryo-

philic behavior, but that their tendency to do so seems to be different.These results suggest that the balanced activity of HSF-1 is importantfor proper thermotactic response.

The thermotaxis defect in hsf-1(sy441) mutants (hereafter referred

to as hsf-1 mutants) might simply reflect their temperature-sensitivedebility19, thus causing limited migration ability for all sensory cues

at 23 C. We therefore addressed whether other sensory responses,such as odorant chemotaxis20,21 ofhsf-1 mutants cultivated at 23 C,

are more defective than those cultivated at 17 C (Suppary

Fi. 3). The hsf-1 mutants cultivated at 23 C did not exhibit

olfactory defects of greater severity than the 17 C-cultivated mutants.Moreover, hsf-1 mutants cultivated at 23 C moved freely like wild-

type worms on the thermotaxis plate without a thermal gradient

a

b

e f

i

c

Cultivated

at 23 C

4.00

Linear TTX assay

Microarray

Linear TTX assay

Microarray

0 1 2 3

Time (h)

4 5 6

Shift

to17

C

at0h

4hafter

shifti

ngto

17C

3.002.001.00

TTXi

ndex

0

14

Intensityat1h(a.u.)

Intensityat2h(a.u.)

Intensity at 0 h (a.u.)

1.002.003.004.00

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10

8

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2 4

HSF-1 binding element

hsp-16.2p

(500 bp)

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40 m

40 m g h40 m 40 m

gfp

8

2 h after transient heat-

shock treatment

10 12 14


Cultivation at 15 C 2 h after shifting to 23 C 2 h after shifting to 25 C

2 4 6 8 10 12 14

d

Intensityat4h(a.u.) 14

12

10

8

6

4

2


2 4 6 8 10 12 14

Figure 1 The essential role o HSF-1 in a

memory-based behavior revealed by genome-

wide microarray analyses. (a) Procedure or

microarray analysis o the thermotactic (TTX)

behavior conditioning process. C. elegans

were cultivated at 23 C or 2.5 d and were

then shited to a new temperature o 17 C.

Immediately ater start o the assay, we isolated

mRNA rom worms that were not used or the

behavioral assays. Error bars represent s.e.m.

(bd) Overall transition o gene expression

change during behavioral conditioning.

Scatter plot o intensity values (log2) or a

representative hybridization o RNA isolated

rom worms at 1 h (b), 2 h (c) or 4 h (d) ater

shiting the cultivation temperature rom

23 C to 17 C compared with that isolated

rom worms just beore shiting the cultivation

temperature. A.u., arbitrary units. (ei) The

HSF-1 activity within a range o cultivation

temperatures in vivo. The hsp-16.2p::gfp

reporter gene, including the two HSF-1 binding

elements (HSEs), was expressed in a wild-type

background (e). Each worm was cultivated

with ood at 15 C or 7 d. The GFPobservations were perormed beore shiting

the temperature (f) and at 2 h ater shiting

to the new temperatures (which were within

the range o normal cultivation temperatures);

23 C (g) or 25 C (h). For the transient heat-

shock treatment (i), each worm was incubated at 35 C or 15 min and then allowed to recover at 15 C or 2 h beore the

GFP observation. The GFP observations were conducted at least our times in each experimental condition.


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(Suppary Fi. 5bf). These results suggest that the abnormal

thermotactic behavior ofhsf-1 mutants does not simply arise fromtemperature-sensitive debility but instead reflects a defect in the

mechanism specific for thermotaxis.To examine whether HSF-1 acts after a temperature shift from 17 C

to 23 C, we constructed worms expressing in a wild-type backgroundan HSF-1 dominant negative form (HSF-1DN)22 under the hsp-16.2

promoter and induced expression of HSF-1DN by heat-shock treat-

ment after worms had reached the adult stage. As the activity of thehsp-16.2 promoter itself is regulated by HSF-1, we co-injected hsp-

16.2p::gfp to confirm whether expression of HSF-1DN appropriately

inhibits the endogenous HSF-1 activity by monitoring the GFP fluo-rescence from this reporter gene. Just before heat-shock treatment,

we observed hardly any fluorescence from worms carrying only thereporter gene and from those carrying hsp-16.2p::hsf-1dn and the

reporter gene, and showed that both groups appropriately migratedtoward lower temperature regions of around 17 C on the thermal

gradient (data not shown). The wild-type worms carrying only thereporter gene and those carrying both hsp-16.2p::hsf-1dn and the

reporter gene were then subjected to the transient heat-shock treat-ment just before a temperature shift from 17 C to 23 C. At 3 h after

a temperature shift from 17 C to 23 following the transient heat-

shock treatment, wild-type worms carrying both hsp-16.2::hsf-1dnand the reporter gene showed slightly less fluorescence than wild-typeworms carrying only the reporter gene (Suppary Fi. 6), sug-

gesting that HSF-1DN inhibited the activity of endogenous HSF-1.

Moreover, wild-type worms carrying both hsp-16.2::hsf-1dn and thereporter gene showed abnormal cryophilic behavior compared with

wild-type worms without both hsp16.2::hsf-1dn and the reporter gene(data not shown) and compared to those carrying only the reporter

gene (Fi. 2). These results suggest that HSF-1 functions after shift-ing the temperature from 17 C to 23 C. The thermotactic defects

characteristic of hsf-1 mutants suggest that HSF-1 is required forresponding to higher temperature, for memorizing higher tempera-

ture or for both processes.

Importance of HSF-1 downstream signaling in thermotaxis

Notably, thermotactic behavior was barely affected by the tran-sient heat-shock treatment, although as expected markedly affected

by shifting the cultivation temperature (Fi. 3a). To identify thegenes involved in the HSF-1 downstream signaling that is involved

in regulating thermotaxis, we classified the genes that were dif-ferentially expressed under microarray analyses into functional

categories (Suppary Fi. 1b) and then explored whether

expression changes of representative genes in each category areHSF-1-dependent.After shifting the temperature from 15 C to 25 C, or vice

versa, amounts of mRNA for hsp genes (Suppary Fi. 3)were substantially changed in wild-type worms, whereas changes

in mRNA amounts were smaller in hsf-1 mutants. Similarly, theHSF-1-dependent expression changes were observed for genes

encoding proteins that potentially have catalytic ac tivity, such asDHS-4 (hydroxysteroid 17 dehydrogenase 11) (Fi. 3b); genes

encoding ion and peptide transporters, such as PGP-9 (ABC trans-porter superfamily) (Fi. 3); and other gene functions (Fi. 3).

Thus, cultivation temperaturedependent changes in expression ofgenes with certain funct ions seem to occur in an HSF-1-dependent

manner. Consistent with the importance of HSF-1 in thermotaxis,

mutants for kgb-2 (Jun N-terminal kinase), dhs-4, kqt-2 (KvQLT-family potassium channel),pgp-9 and max-1 (a regulator of axonguidance) showed abnormal thermotactic migration (Fi. 3). By

contrast, the amounts of mRNA for genes such as cgt-1 (ceramideglucosyltransferase), pmp-1 (long-chain acyl-CoA transporter)and dmd-7 (the transcription factor doublesex) changed inde-

pendently of HSF-1 (Fi. 3b). Mutants for these genes showedalmost normal thermotaxis (Fi. 3). Thus, quantitative polymer-

ase chain reaction (PCR) analysis and behavioral analysis sug-gest that expression of several genes, such as dhs-4, is directly

or indirectly regulated by HSF-1, and that HSF-1-dependentchanges in downstream signaling are likely to be important

for thermotaxis.

Figure 2 Characterization o thermotaxis

behavior o hsf-1 mutants. (a,b) Population

thermotaxis (TTX) temperature shit assay

o wild-type worms, hsf-1(sy441)mutants19

and hsf-1-overexpressing worms10.

Cultivation temperature was shited rom

17 C to 23 C (a) and vice versa (b). The

population TTX assay was conducted just

beore shiting the cultivation temperature

(0 h) and at every 1 h ater shiting

the temperature. (c) Thermotaxis o

hsf-1(ok600)null mutants. Population

TTX assays were conducted at 2 h ater

shiting the temperature rom 17 C to

23 C. **P< 0.01 (t-tests, n = 3).

(d) Evaluation o the eect o long-

term cultivation at 23 C ater shiting

temperature, on the thermotaxis o hsf-1

mutants and hsf-1-overexpressing worms.

Population TTX assays were conducted at

7 h and 10 h ater shiting temperature

rom 17 C to 23 C. *P< 0.05,

**P< 0.01 (t-tests, n = 3). (e) Assessment

o the dominant negative eect o HSF-1 on

thermotaxis in the adult stage o wild-typeworms. HSF-1DN was expressed in wild-type

worms under hsp-16.2promoter (hsp-16.2p::hsf-1dn). Population TTX assays were perormed at 3 h ater heat-shock treatment ollowed

by the temperature shit rom 17 C to 23 C. **P< 0.01 (t-tests, n = 3). NS, not signiicant. Error bars represent s.e.m.

3.00

4.00

3.00

2.00

1.00

0

2.00

1.00

0

TTXi

ndex

TTXi

ndex

TTXi

ndex

TTXi

ndex

TTXi

ndex

3.00

1.00

4.00

3.00

2.00

1.00

0

7 h after shifting

to 23 C

10 h after shifting

to 23 C

1.00

3.00

2.00

1.00

0

1.00

12

Time (h)

Time (h)

Wild typehsf-1(sy441) mutanthsf-1 overexpression

Wild type

hsf-1(sy441)mutant

hsf-1 overexpression

3 4 5

1 2 3 4 5

2.00

1.00

3.00

2.00

1.00

0

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2.00

1.00

NS NS NS

**

**

*

**

**

****

a

c d e

b

hsf-1

(ok6

00)

hsf-1

(sy4

41)

Wild

type

hsf-1

over-

expre

ssion

hsf-1

(sy4

41)

Wild

type

hsf-1

over-

expre

ssion

hsf-1

(sy4

41)

Wild

type

Wild

type

hsf-1

dn


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calcium transients of AFD and AWC in the hsf-1 mutants by calcium

imaging with a genetically encoded calcium indicator, cameleon.When temperature was raised and then lowered, the fluorescence

resonance energy transfer (FRET) ratios in AFD changed less in hsf-1mutants than in wild-type worms (Fi. 6a). By contrast, AWC in hsf-1

mutants was more responsive to the transient temperature changefrom 17 C to 23 C than AWC in wild-type worms (Fi. 6b). These

results suggest that the HSF-1 signaling is involved in modulation ofthe responsiveness of AFD and AWC to the transient temperature

change. Consistent with these observations, FRET ratios of the AIY

interneuron, postsynaptic to both AFD and AWC, in hsf-1 mutantswere substantially less than those in wild-type worms, probablyowing to the decreased and increased responsiveness of AFD and

AWC to transient temperature changes in hsf-1 mutants, respectively

(Suppary Fi. 9). Expression ofhsf-1 in body wall musclesrestores the temperature responsiveness of AFD, AWC and AIY inhsf-1 mutants (Fi. 6a,b and Suppary Fi. 9b). These resultsare consistent with the results from genetic epistasis analysis, which

found that the HSF-1 signaling regulated AFD and AWC (Fi. 5).Previous calcium imaging experiments have shown that, when tem-

perature is raised from 15 C to 25 C in a step-wise manner, AFDand AWC neurons respond to every warming above a threshold tem-

perature that is set by the previous cultivation temperature, indicating

a

300 m

e f

g

i

h

50 m

Nerve ringb

30 m

Body wall musclec

40 m

Intestined

3.00

Wildtype

hsf-1

unc-14p

Wildtype

Pan-neuronal

hsf-1

unc-14p

***

3.00

2.00

2.00

1.00

1 ng l1

10 ng l1

TTXindex

1.00

0

3.00

Wildtype

hsf-1

myo-3p

Wildtype

Body wall

muscles

hsf-1

myo-3p

Wildtype

hsf-1

ges-1p

Wildtype

hsf-1

ges-1p

Wildtype

hsf-1

ges-1p::gfp

** ** **

3.00

2.00

2.00

1.00

1 ng l1

10 ng l1

Intestine Marker

gene

1 ng l1

10 ng l1

50 ng l1

TTXindex

1.00

0

3.00

Wildtype

hsf-1

gcy-8p,ceh-36p,

AIZp,g

lr-3p

Thermotactic

neural circuit

**

3.00

2.00

2.00

1.00

TTXindex

1.00

0

Wildtype

hsf-1

ttx-3p

AIY AIZ RIA

3.00

3.00

2.00

2.00

1.00

TTXindex

1.00

0

* ***

1 ng l1

Wildtype

hsf-1

ttx-3p

10 ng l1

10 ng l1

10 ng l1

Wildtype

hsf-1

lin-11p

Wildtype

hsf-1

lin-11p

1 ng l1

Wildtype

hsf-1

glr-3p

Wildtype

hsf-1

glr-3p

1 ng l1

3.00

Wildtype

hsf-1

gcy-8p

Wildtype

hsf-1

gcy-8p

Wildtype

hsf-1

ceh-36p

Wildtype

hsf-1

ceh-36p

Wildtype

hsf-1

sra-6p

** **

3.00

2.00

2.00

1.00

TTXindex

1.00

0

AFD AWC ASH

1 ng l1

10 ng l1

1 ng l1

10 ng l1

1 ng l1

Wildtype

hsf-1

sra-6p

Wildtype

hsf-1

gcy-5p

gcy-7p

Wildtype

hsf-1

gcy-5p

gcy-7p

10 ng l1

ASE

1 ng l1

10 ng l1

Figure 4 Cell-speciic rescue o deective

thermotactic behavior in hsf-1 mutants.

(ad) Expression pattern o HSF-1 in wild-type

worms. To drive the GFP luorescence under the

hsf-1 promoter, hsf-1 promoter::gfpreporter

gene was injected into the wild-type worms.

The GFP luorescence was detected in nearly

all cells (a): or example, in the nerve ring (b),

body wall muscles (c) and intestine (d).

(ei) Cell-speciic rescue experiments.

Thermotactic behavior was examined at 2 h

ater shiting temperature rom 17 C to 23 C

by population thermotaxis (TTX) assays.

hsf-1 cDNA was expressed as a transgene

(1 ng l1 or 10 ng l1) in almost all neurons

(pan-neuronal; e or in the body wall muscles

or intestine (f) o hsf-1 mutants using individual

cell-speciic promoters with ges-1p::gfp

(50 ng l1) as an injection marker.

The transgene was also coordinately expressed

in AFD neurons at 1 ng l1, AWC neurons

at 10 ng l1 and AIZ and RIA neurons at

1 ng l1 (g) and individually expressed in the

sensory neurons AFD, AWC, ASH or ASE (h) or

in the interneurons AIY, AIZ or RIA ( i). At leasttwo lines were assayed or each transgenic

strain. Experiments or each transgenic strain

were carried out at least three times. The

signiicant dierences o thermotactic plasticity

between each transgenic strain and hsf-1(sy441)

mutants were determined by Fishers protected

least signiicant dierence multiple-comparison

test. *P< 0.05, **P< 0.01. Error bars

represent s.e.m.

genetic epistasis analysis suggests that the HSF-1-mediated non-cell-autonomous signaling regulates AFD and AWC thermosensory

neurons, AIY interneurons or both the thermosensory neurons and

interneurons, possibly through its effects downstream of the cGMP-dependent pathway (Suppary Fi. 7).Although recent studies addressed whether the HSF-1 activation is

dependent on the AFD neuron16 or not27, our quantitative PCR analy-sis suggests that expression changes of the hsp genes in response to

both heat-shock stimuli and cultivation temperature changes were notsubstantially different between wild-type worms andgcy-23 gcy-8 gcy-18 triple mutants, or ttx-3 mutants (Suppary Fi. 8). We alsoshowed that the GFP fluorescence level of non-neuronal cells, such as

intestine ingcy-23 gcy-8 gcy-18 mutants or ttx-3 mutants expressinghsp-16.2 reporter constructs, is comparable to the fluorescence of

those in wild-type worms. The results in this study were consistentwith a recently reported result suggesting that the HSF-1 activity is

independent of the ttx-1 mutation that is known to specifically disrupt

AFD structure27, but partly contradicted a previous study16 that foundthe AFD and AIY neurons to be important for the expression of a fewhsp genes. This contradiction might arise from the subtle difference in

growth conditions. In particular, humidity has been known to affecttemperature sensation and thermotaxis, although little is known about

the mechanism dictating the relationship between hygrosensation andthermosensation. Taken together, at least in our growth condition,

expression of a few HSF-1 downstream genes such as hsp-70 seems to

be independent ofgcy-23 gcy-8 gcy-18 and ttx-3 mutations.

Regulation of the thermosensory neurons by HSF-1 signaling

To address the possibility that the HSF-1 signaling affects AFD and

AWC sensory neurons, we monitored temperature stimulusevoked


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that these thermosensory neurons can, of themselves, memorize theperceived cultivation temperature9,28. To investigate the responsive

temperature range, we also measured the FRET ratio changes of AFDneurons of wild-type worms and hsf-1 mutants during the step-wise

warming (Fi. 6). Consistent with the results from the previous cal-cium imaging experiments28, the AFD neuron of wild-type worms

cultivated at 23 C showed a marked response when warmed to atemperature above 23 C (Fi. 6). By contrast, the AFD neuron ofhsf-1 mutants cultivated at 23 C showed a marked response when

warmed to a temperature above 21 C; that is, the response ofhsf-1mutants to warming occurred earlier than that of wild-type worms(Fi. 6,). There was, however, hardly any observable difference in

the cultivation-temperature dependency of AWC response betweenwild-type worms and hsf-1 mutants (Fi. 6). Furthermore, expres-

sion ofhsf-1 in body wall muscles sufficiently rescued an abnormalresponse of the AFD neuron ofhsf-1 mutants (Fi. 6,). These results

indicate that the HSF-1 signaling is important for the cultivation tem-peraturedependent response of the AFD neuron.

Estrogen signaling acts downstream of HSF-1 signaling

We also addressed how the HSF-1 signaling regulates the thermo-sensory neurons. Among genes likely to act downstream of HSF-1,

dhs-4 and cyp-37B1 genes encode hydroxysteroid 17 dehydroge-

nase 11 (Wormbase gene ID, WBGene00000968) and a cytochromeP450 CYP4/CYP19/CYP26 subfamily protein (WBGene00009226),respectively, both of which are believed to be involved in estrogen

synthesis. In a local a lignment search using the sequences ofHomosapiens estradiol 17 dehydrogenase 1 (HSD17B1) and Homo sapiens

cytochrome P450 19A1, known as aromatase (CYP19A1) as queriesagainst the C. elegans genome database, both DHS-4 (25% identity,expected value (E-value) = 2 1021) and CYP-37B1 (23% identity,E-value = 3 109) were ranked in the top five matches. Recentreports have shown that estrogen and its receptor are involved in

hippocampal synaptic plasticity and memory through the regulationof calcium influx2931. We therefore assessed the effect of estrogen

signaling on C. elegans thermotaxis.

We exogenously applied 17-estradiol (referred to as estradiol fromhere on), a main form of estrogen, to wild-type worms grown at 23 C

from their birth. An application of estradiol caused thermophilicmovement in a dose-dependent manner (Fi. 7a). Further, estradiol

application on 23 Ccultivated hsf-1 mutants and dhs-4 mutants par-tially restored their behavioral defects (Fi. 7b,). Notably, estradiol

application only during the behavioral conditioning process causedthermophilic movement of wild-type worms and partially rescued

the behavioral defect ofhsf-1 mutants and dhs-4 mutants (Fi. 7

and Suppary Fi. 1). Despite the clearly observable effectsof estradiol on wild-type worms as well as hsf-1 mutants and dhs-4mutants, estradiol application to ttx-3 mutants did not induce any

obvious effect, suggesting that ttx-3 is genetically epistatic to estrogensignaling as well as to the HSF-1 signaling (Fi. 5, and 7b).

On the basis of the results from the estradiol application experi-ments, we investigated the relationship between HSF-1 signaling and

estrogen signaling. The previous in vitro binding analysis showedthat the three nuclear receptors NHR-14, NHR-69 and NHR-121,

all of which were identified by their highest homologies to humanestrogen receptor, bound estradiol in a dose-dependent fashion32.

We analyzed thermotaxis of mutants impaired in the nhr-14, nhr-69or nhr-121 genes. nhr-69 mutants cultivated at 23 C showed a sub-

stantial tendency to migrate toward colder regions than wild-type

worms, whereas nhr-14 mutants and nhr-121 mutants cultivated at17 C or 23 C showed no obvious defect (Fi. 8a). We further inves-tigated the functional redundancy between NHR-69 and NHR-14 or

NHR-121, examining thermotaxis ofnhr-69;nhr-121 mutants, nhr-69;nhr-14 mutants and nhr-69;nhr-121;nhr-14 mutants. When cul-tivated at 23 C, the behavioral defects in nhr-69;nhr-121 mutants,nhr-69;nhr-14 mutants and nhr-69;nhr-121;nhr-14mutants were eachsimilar to that ofnhr-69 single mutants (Fi. 8a), suggesting that

there is no redundancy between nhr-69 and the other two nhrgenesunder the 23 C-cultivated condition. By contrast, nhr-69;nhr-121

mutants, nhr-69;nhr-14 mutants and nhr-69;nhr-121;nhr-14 mutantseach cultivated at 17 C exhibited a slightly more severe behavioral

defect than 17 C-cultivated nhr-69 mutants (Fi. 8a), suggesting that

b ca

d4.00

TTXi

ndex

3.00

2.00

1.00

0

1.00

2.00

3.00

NS

NS

Wild type hsf-1 odr-3

**

******

**

ttx-3 hsf-1;ttx-3

gcy-23

gcy-8

gcy-18

hsf-1;

gcy-23

gcy-8

gcy-18

hsf-1;gcy-23gcy-8

gcy-18Ex[myo-3p::

hsf-1]

hsf-1;

odr-3

0.50

0.40

0.30

0.20

0.10

0

4 3 2 1 +1 +2 +3 +4

hsf-1

hsf-1;gcy-23

gcy-8 gcy-18hsf-1;gcy-23 gcy-8 gcy-18

Ex[myo-3p::hsf-1]

gcy-23 gcy-8 gcy-18

Wild type

Region

Fractionofanimals

ineachregion

4 3 2 1 +1 +2 +3 +4

0.50

0.40

0.30

0.20

0.10

0

Wild type

ttx-3

hsf-1

hsf-1;ttx-3

Fractionofanimals

ineachregion

0.50

0.40

0.30

0.20

0.10

4 3 2 1 +1 +2 +3 +4

0

Wild type

odr-3

hsf-1

hsf-1;odr-3

Region

Fractionofanimals

ineachregion

Figure 5 Genetic interactions between HSF-1 signaling

and the genes that act in the thermotactic neural circuit.

(ac) Genetic epistasis analysis. Thermotactic plasticity

o wild-type worms and each mutant was examined using

population thermotaxis (TTX) assays at 2 h ater shiting

the temperature rom 17 C to 23 C. The TTX plate

was divided into eight regions, 4 (coldest) to +4

(warmest) (Supplementary Fig. 1a). Epistatic relations

between hsf-1 mutants and gcy-23 gcy-8 gcy-18

mutants (a), odr-3mutants (b) or ttx-3mutants (c) are

shown. The ttx-7;hsf-1 double mutants were also

constructed, but it was diicult to analyze their

thermotactic behaviors owing to their severe locomotory

deects. (d) Summary o genetic epistasis analysis or the relationship between systemic temperature perception mechanism and thermotactic neuralcircuit. Thermotactic plasticity was examined as above. The signiicance o dierences o thermotactic plasticity was determined using the Fishers

protected least signiicant dierence multiple-comparison test. **P< 0.01; NS, not signiicant. Error bars represent s.e.m.


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a r t I C l e S

NHR-69 functions redundantly with NHR-14 and NHR-121 under

the 17 C-cultivated condition. Taken together, these data suggest that

at least NHR-69 is required for thermotaxis.We then conducted a genetic epistasis analysis between nhr-69 andhsf-1. The behavior of nhr-69 hsf-1 double mutants was similar to

that ofnhr-69 mutants (Fi. 8b), indicating that nhr-69 is epistatic tohsf-1. This result suggests that NHR-69, which might mediate both

thermophilic and cryophilic movements, could act downstream of theHSF-1 signaling to be involved in the former movement. To further

address this possibility, we took a pharmacological approach to testwhether estradiol application affects thermotaxis ofnhr-69 mutants.

Estradiol application had no substantial effect on the thermotacticbehavior ofnhr-69 mutants (Fi. 8,). Considering the effect of estra-

diol application on hsf-1 mutants (Fi. 7b), this result is consistentwith the idea that NHR-69 acts downstream of the HSF-1 signaling.

Given that ttx-3 is epistatic to estrogen signaling (Fi. 7b),

we further attempted to identify a site of action of NHR-69. We

examined whether the thermotactic behavior ofnhr-69 hsf-1 doublemutants was restored to that ofhsf-1 single mutants when NHR-69is expressed in AFD, AWC or AIY neurons ofnhr-69 hsf-1 mutants.

The expression of nhr-69 cDNA in AWC or AIY neurons had noobvious effects on the behavior ofnhr-69 hsf-1 mutants, but strong

expression ofnhr-69 (3 ng l1) in the AFD neuron of nhr-69 hsf-1mutants caused a strong tendency to migrate toward colder regions

rather than a restoration to the phenotype of hsf-1 single mutants(Fi. 8b). These results suggest that NHR-69 acts in AFD, although

excess expression ofnhr-69 in AFD on the HSF-1 signalingreducedbackground leads to an increase in inactive NHR-69 in the AFD,

which might cause a stronger tendency for nhr-69 hsf-1 mutantsto migrate to colder regions. Indeed, weaker expression of nhr-69

a b c

e

d

24

Ratiochange(%)

AFD neuron

23 C

17 C 17 C

Wild typehsf-1(sy441)hsf-1(sy441);Ex

[myo-3p::hsf-1]18

12

6

0

6

12

0 30 60 90 120

Time (s)

150 180 210 240

Ratiochange(%)

AFD neuron

15 C17 C

19 C21 C

23 C25 C

30

0 30 60 90 120

Time (s)

150 180 210 240

20100

20100

20100

AWC neuron

15 C17 C

19 C21 C

23 C25 C

0 30 60 90 120

Time (s)

150 180 210 240

Ratiochange(%)

302010

02010

02010

0

Wild

type

hsf-1

(sy4

41)

hsf-1

(sy4

41);

Ex[m

yo-3p::hsf-

1]

Ratiochange(%)

12NS

** **

9

6

3

0

AWC neuron

23 C

17 C 17 C

12

Ratiochange(%)

8

4

0

4

8

0 30 60 90 120

Time (s)

150 180 210 240

Wild typehsf-1(sy441)hsf-1(sy441);Ex

[myo-3p::hsf-1]

Figure 6 Regulation o the thermosensory neurons by the HSF-1 signaling. (a,b) In vivocalcium

imaging o the thermosensory neurons in 23 C-conditioned wild-type worms, hsf-1 mutants

and hsf-1 mutants expressing hsf-1 in body wall muscles during transient up-and-down warming.

This method o warming was used to examine calcium transients in AFD (a) and AWC (b). The average ratio changes o

AFD and AWC were measured or wild-type worms (blue), hsf-1 mutants (red) and hsf-1 mutants expressing hsf-1 inbody wall muscles (green). Stimulus temperatures are shown at the bottom. n = 11 to 17 worms. (ce) In vivocalcium

imaging o the thermosensory neurons in 23 C-conditioned wild-type worms, hsf-1 mutants and hsf-1 rescue lines

during step-wise warming. Temperature was increased in a step-wise manner to examine the temperature memory o

the AFD (c) and AWC (d) thermosensory neurons, which was set by the previous cultivation temperature. The average

ratio changes in AFD (c) and AWC (d) were measured each or wild-type worms (blue), hsf-1 mutants (red) and hsf-1

mutants expressing hsf-1 in body wall muscles (green). Arrows indicate the irst marked ratio changes during s tep-

wise warming. The average ratio change o the AFD neurons in response to the temperature change rom 19 C to 21 C

during step-wise warming is shown in a bar graph (e). **P< 0.01; NS, not signiicant (t-tests, n = 12 to 20 worms).

Error bars represent s.e.m.

3.00a

2.00

** **

** **

1.00

TTXi

ndex

0

1.00Concentration (M)

108

107

106

105

104

103

4.00b3.00

3.00

2.00

2.00

***

**

1.00

1.00TTXi

ndex

0

4.00

OffWild type h sf -1 d hs -4

ttx-3

NS

On Off On Off On

Off On

4.00d3.002.00

2.00

****

**

1.00

1.00TTXi

ndex

0

3.00

Off

Wild type h sf -1 d hs -4

ttx-3

NS

NS

NSNSNS

On Off On Off OnOff On

Off

Wild type h sf -1 d hs -4

On Off On Off On1.00

c

2.003.00

1.00

TTXi

ndex 0

4.00

ttx-3

Off On

Figure 7 Eect o estradiol application on thermotaxis. (a) The dose-

dependent eect o estradiol application. Wild-type worms were cultivated

at 23 C with indicated concentration o estradiol. To clearly observe the

thermophilic movement o worms cultivated at 23 C, the center o the

thermotaxis (TTX) plate was adjusted to 23 C to establish a linear thermal

gradient ranging rom approximately 20 C to 26 C. However, other behavioral

assays were conducted by adjusting the center o the TTX plate to 20 C

to establish a thermal gradient ranging rom approximately 17 C to 23 C

(see Supplementary Fig. 1). (b,c) Eect o exogenously applied estrogen on

thermotactic behaviors o wild-type worms and several mutants. Each worm

was cultivated at 23 C (b) or 17 C (c), either with (on) or without (o) 10 M

estradiol. (d) Eect o estradiol on the behavioral conditioning process.

Assay was conducted as described in Supplementary Figure 1c. *P< 0.05,

**P< 0.01 (t-tests). Error bars represent s.e.m.


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a r t I C l e S

(0.3 ng l1) in AFD restored the thermo-

tactic behavior ofnhr-69 hsf-1 double mutants

(Fi. 8b). To further examine whether theexpression ofnhr-69 in AFD affects the AFD

activity in 23 C-cultivated nhr-69 hsf-1mutants, we measured calcium transients

of the AFD in nhr-69 hsf-1 mutants and themutants expressing nhr-69 cDNA in the AFD

at 0.3 ng l1 or 3 ng l1. Consistent withthe results from behavioral assays (Fi. 8b),

expression of nhr-69 in the AFD neuron of nhr-69 hsf-1 mutants

affected the AFD activity in a dose-dependent manner (Fi. 8).Together with the results from the exogenous estrogen application,genetic and calcium imaging analyses, this suggests that HSF-1 sig-

naling acts at least in part through estrogen signaling, directly orindirectly, to regulate the thermosensory neuron AFD.

DISCUSSION

In the current study, we found that systemic temperature signal-ing regulates the thermosensory neurons for thermotaxis through

heat-shock transcription factor and estrogen signaling. The heat-shock transcription factor has been well known as a thermosensor

that is highly conserved from organisms as distant as Saccharomyces

cerevisiae. By contrast, the AFD neuron is a specialized thermosensor

with sensitivity enough to respond to differences of 0.01 C on a

thermal gradient8. We therefore infer that C. elegans added its finetemperature sensitivity through the evolution of the AFD thermosen-sory system. Given that the HSF-1 protein responds to temperature

through its multimerization ability, it is unlikely that HSF-1 is as sen-

sitive as AFD. Alternatively, as a plausible purpose of the temperatureresponse, HSF-1 might function during behavioral conditioning after

temperature shifts from 17 C to 23 C in an incubator, as microarrayanalysis, quantitative PCR analysis and GFP observation indeed indi-

cated that expression of several genes downstream of HSF-1 changedduring this process (Fis. 1h and 3b and Suppary Fis. 2

and 3a,b,). Although the AFD neurons can respond to temperaturedifferences of 0.01 C for isothermal tracking, worms were broadly

distributed around the cultivation temperature in the temperature

gradient in population assays (Fi. 5a). It is probable that C. elegans

possesses multiple types of thermosensory systems that are not mutu-ally exclusive.Given the recent studies indicating that the sensory system

operating behavior is more diverse than previously thought33, it isreasonable to consider the possibility that animals can perceive envi-

ronmental changes not only through a specialized sensory systembut also through sensors distributed across the body. For example, a

small set of warmth-activated anterior cell neurons is located in theinternal portion of the Drosophila melanogasterbrain, whose func-

tion is important for preferred temperature selection34. In addition,D. melanogaster larvae perceive light by using neurons embedded

under the cuticle encasing their bodies35. This sensory system doesnot eliminate the importance of photoreceptors in eyes. In C. elegans,

the PVD neurons that are located on either side of the worm and

that envelop the body with multidendritic processes respond to coldtemperatures36. Thus, it is possible to suppose that an HSF-1-mediatedmechanism might be responding to a signal from other neurons,

such as PVD neurons. Although further work is needed to clarify

the relationship among the various temperature signaling systems,this study and others3336 suggest that sensory systems influencing a

behavior might be broadly required throughout an animals body.A principal view in neuroscience is that environmental temperature

is detected by sensory neurons in the nervous system. We demon-strate here that temperature signaling by non-neuronal cells triggers

behavioral changes, as thermosensory neurons do. Given the robustinfluence of temperature on animals bodies, it improves behavioral

performance to monitor environmental temperatures at the level of

b

c e

d

NS

NSNS

NS

NS

NS

NS

****

3 0.3

gcy-8p ceh-36p ttx-3p

ng l1

:

nhr-69 hsf-1 worms expressingpromoter::nhr-69 cDNA

0.03 3 3

**

4.00

3.00

2.00

1.00

0

1.00

2.00

60 AFD neuronWild type

nhr-69 hsf-1

nhr-69 hsf-1 wormsexpressinggcy-8p::nhr-69 (0.3 ng l

1)

nhr-69 hsf-1 worms expressing

gcy-8p::nhr-69 (3 ng l1

)

50

40

30

Ratiocha

nge(%)

20

10

0

0 30

17 C 17 C

23 C

60 90 120

Time (s)

150 180 210 240

4.00

Off On

Wild type

Wild type

nhr-69

nhr-69

Off On

Off On Off On

3.00

2.00

1.00

0

0

1.00

2.00

3.00

TTXi

ndex

TTXi

ndex

TTXi

ndex

a NS

NS

NS

NS

NS

** **

** ** **

****

3.00

2.00

1.00

0

0

1.00

2.00

3.00

TTXi

ndex

TTXi

ndex

Wild

type

nhr-6

9

hsf-1

nhr-6

9hs

f-1

nhr-1

4

nhr-6

9

nhr-1

21

nhr-69

;nhr

-14

nhr-6

9;nhr

-121

nhr-69

;nhr

-121

;

nhr-1

4

Figure 8 HSF-1 signaling acts through estrogen

signaling to regulate the AFD thermosensory

neurons. (a) The thermotactic (TTX) behavior

o the mutants impaired in the putative

estrogen receptors. nhr-14(tm1473)mutants,

nhr-69(tm2365)mutants, nhr-121(tm1797)

mutants and their double or triple mutants were

cultivated at 23 C or 17 C and then assayed.

(b) Genetic interactions between hsf-1 and

nhr-69. Thermotaxis o worms was examined

2 h ater shiting temperature rom 17 C

to 23 C. Concentrations o nhr-69cDNA

expressed in nhr-69 hsf-1 mutants under cell-

speciic promoter were indicated. (c,d) Eect

o estradiol on thermotaxis o nhr-69mutants.

Worms were grown at 23 C (c) or 17 C (d) with

or without 100 M o estradiol. (e) Calcium

imaging o AFD neurons in 23 C-conditioned

wild-type worms (n = 9), nhr-69 hsf-1 mutants

(n = 12) and nhr-69 hsf-1 mutants expressing

nhr-69in AFD neurons at the concentration

o 0.3 ng l1 (n = 15) or 3 ng l1 (n = 13).

**P< 0.01; NS, not signiicant (t-tests). Error

bars represent s.e.m.


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a r t I C l e S

the entire body. To ensure that this sort of strategy is retained throughevolution, heat-shock factor, a highly conserved protein, might adopt

a role in transcriptionally coupling environmental recognition to amemory-regulated behavior, such as thermotaxis in C. elegans.

METHODS

Methods and any associated references are available in the online

version of the paper at http://www.nature.com/natureneuroscience/.

Assi s. Gene Expression Omnibus: Microarray data have

been deposited with accession code GSE28856.

Note: Supplementary information is available on the Nature Neuroscience website.

AcknowledgmentS

We thank N. Hisamoto (Nagoya University), T. Mizuno (Nagoya University)and K. Matsumoto (Nagoya University) for sharing strains; A. Fire (StanfordUniversity School of Medicine) for pPD plasmids; Y. Iino (University of Tokyo)for thegcy-5 andgcy-7promoters; Caenorhabditis Genetic Center, C. elegansKnockout Consortium and S. Mitani at the National Bioresource Project, Japan,for strains; K. Terauchi and T. Kondo for kindly sharing the microarray apparatus;T. Inada for kindly sharing the quantitative PCR apparatus; C. Bargmann,S. Takagi, N. Hisamoto, A. Kuhara, P. Jurado, H. Sasakura, N. Ohnishi, T. Kimata,

M. Nonomura and members of the Mori laboratory for comments on thismanuscript and discussion. I.M. is a Scholar of the Institute for Advanced Researchin Nagoya University, Japan. This work was supported by the Core Research forEvolutional Science and Technology (CREST) Program of the Japan Science andTechnology (JST) agency (to I.M.).

AUtHoR contRIBUtIonS

T.S. designed the research, performed most experiments, analyzed data and wrotethe manuscript; Y.N. performed the quantitative PCR experiments and conductedgermline transformation to construct the C. elegans transgenic line; I.M. supervisedthe project, conducted initial identification of cells expressing the hsf-1 promoter::gfpreporter gene and wrote the manuscript.

comPetIng FInAncIAl InteReStS

The authors declare no competing financial interests.

Published online at http://www.nature.com/natureneuroscience/.Reprints and permissions information is available online at http://www.nature.com/

reprints/index.html.

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nature neurOSCIenCedoi:10.1038/nn.2854

ONLINE METHODSgis. The hsf-1(sy441) mutation19 was outcrossed to the wild type six times.

Thesy441 mutant has a single G-to-A mutation in the seventh exon (5-AGT AAT

CAT AAT TGG GAT GAT TTT GGG AAT-3), which would convert residue

585 (tryptophan) to a stop codon, leading to the truncation of the last 86 amino

acids at the C terminus. Double, triple and quadruple mutants in this study were

constructed by genetic and molecular methods. hsf-1(ok600) is a null allele and

is kept as a balanced heterozygous strain.

gri rasfrai. Germline transformations were performed

as described37. For cell-specific rescue experiments, co-injection mixes

consisted of pKDK66ges-1p::NLS GFP (50 ng l1) and experimental DNA

at various concentrations (1 or 10 ng l1). To analyze the expression

pattern of HSF-1, pTAK16 hsf-1p::gfp (20 ng l1) were injected into wild-

type worms. For GFP observation, pTAN51 hsp-16.2p::gfp (50 ng l1)

and pTAK14 hsp-16.2p(hsemut)::gfp (50 ng l1) were co-injected with pNAS88

ges-1p::NLS TaqRFP(50 ng l1) and pTAN124ges-1p::NLS DsRed monomer

(50 ng l1), respectively.

RnA isai a apifiai. For synchronization of worm growth38,

worms were initially grown to starvation in a 60-mm plate of nematode growth

medium (NGM) at 23 C. Half of the agar in the plate was split into four pieces,

and two pieces were placed on a 150-mm plate. The resultant plates were incu-

bated at 23 C until most of the food was depleted. The worms were collectedfrom the plates using M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 85 mM

NaCl and 1 mM MgSO4) and then resuspended in 75 ml bleach solution (15 ml

sodium hypochlorite (Kao), 3.75 ml 10 M NaOH and 56.25 ml water). Worms

were transferred to a 200-ml glass beaker with a stir bar and incubated for

5 min while stirring rapidly. When most adults had burst, the solution was passed

through a 53-m nylon mesh (Tanaka Sanjiro Co.) to separate intact embryos

from worm debris. Embryos were collected by centrifugation, resuspended in

2 ml M9 buffer and split equally over six 150-mm NGM plates. The plates were

incubated at 23 C for 2.5 d for microarray analysis. The hatched worms were

collected from the plates using S-basal buffer (5.7 mM K2HPO4, 44 mM KH2PO4,

100 mM NaCl and 5 g ml1 cholesterol), and a fraction of worms was used

for the temperature shift thermotaxis assay, whereas the remaining worms were

killed and dissolved in TRI reagent (Ambion) for microarray analysis. mRNA was

isolated before shifting the temperature from 23 C to 17 C (0 h at 23 C) and at

every 1 h after shift ing temperature to 17 C (1, 2, 3 or 4 h at 17 C) (Fi. 1a). TotalRNA was isolated from the lysate using the RiboPure kit (Ambion).

The high-fidelity linear amplification of 40 ng of isolated mRNA was accom-

plished using the WT-Ovation Pico RNA Amplification System (NuGEN)

according to the manufacturers protocol, with some modifications39. The ampli-

fied cDNA was fragmented and biotinylated using FL-Ovation cDNA Biotin

Module V2 (NuGEN).

mirarray. Fragmented, biotin-labeled cDNA (50l) was mixed with hybridi-

zation cocktail (Affymetrix) and hybridized to the Affymetrix C. elegans chip

for 18 h at 45 C, according to the Affymetrix Expression Analysis Technical

Manual. The hybridized arrays were washed and scanned using the GeneChip

Scanner 3000 TG system. The C. elegans chip was designed using the December

2000 genome sequence.

Microarray data were processed as described38,40,41. Primary intensity values

from each hybridization were scaled against a global average signal from the samearray and normalized with Robust Multichip Average analysis (RMA). To identify

differentially expressed transcripts during the worms temperature memory acqui-

sition process, normalized intensity values from gene expression data sets of worms

cultivated for 4 h after shifting the temperature to 17 C were compared with those

from reference gene expression data sets of worms isolated before shifting tempera-

ture, using Significance Analysis of Microarray software (SAM)38,40,41.

Aaysis f irarray aa. A two-class paired analysis of the data was per-

formed to identify genes that differed by1.8-fold from the reference at a false

discovery rate of 1.12% for expression data sets of RNA isolated at 4 h after shift-

ing temperature to 17 C. In this set of arrays, 46 upregulated and 33 downregu-

lated genes (Suppary Fi. 2) were found to be significantly regulated, with

6.813 median false significant genes. The 79 differently expressed genes were

classified according to their gene ontology annotations using GoMiner software

(http://discover.nci.nih.gov/gominer/)40. To validate microarray data, the expres-

sion of some of genes was tested by quantitative PCR.

gFP bsrvai. All observations were carried out using an Axioplan2 light

microscope (Carl Zeiss) except for those in Fiur 4a, in which the fluo-

rescence images were taken with a Fluoview FV1000 confocal laser scanning

microscope (Olympus). Worms expressing reporter genes, hsp-16.2p::gfp or

hsp-16.2p(hsemut)::gfp, were cultivated at 15 C. Adult worms were transferredto the new cultivation temperature of 20 C, 23 C or 25 C, or to the heat-

shock temperature of 30 C and 35 C, and their GFP fluorescence was moni-

tored at 2 h or at each time point as shown in Suppary Fiur 3. The

GFP intensity of each worm was calculated as described in the figure legend

ofSuppary Fiur 3 and was quantified using ImageJ software (US

National Institutes of Health).

thraxis assay. A radial temperature gradient assay was carried out using a

9-cm agar plate and a vial containing frozen acetic acid, according to previously

described methods4,5. A thermotaxis assay using a linear temperature gradient

was also performed as previously reported6. Equipment for establishing the linear

thermal gradient was used as previously described3. A stable, linear thermal

gradient was established on a thin, 60-cm long aluminum platform, one end of

which was placed in a water bath at 5 C, with the opposite end in a water bath

at 35 C. A thermotaxis plate (13.5 cm 6 cm, 1.8 cm height) containing 10 mlof thermotaxis medium (3 g l1 NaCl, 20 g l1 Bacto Agar (Becton Dickinson)

and 25 mM KPO4) was placed on the aluminum platform such that the tem-

perature gradient could be established along the agar surface of 13.5 cm. The

center of the 13.5-cm-long agar surface in the thermotaxis plate was adjusted

to 20 C and the thermotaxis plate was maintained for 15 min, until a linear

thermal gradient ranging from approximately 17 C to 23 C was established.

Worms were grown on a 6-cm plate containing 14 ml of NGM with agar, on

which E. coli OP50 was seeded; each worm and its progeny were cultured at

17 C or 23 C. Worms were collected with 1 ml of S-basal buffer kept at 20 C

and were washed once with autoclaved water at 20 C. These steps were carried

out within 7 min in a water bath with constantly maintained temperature of

20 C. Approximately 80200 worms were placed at the center of the thermo-

taxis plate. Excess water surrounding the worms was removed with tissue paper.

After 60 min, the worms were killed by chloroform gas, and the worms in each

of the eight regions were scored. The thermotaxis index was calculated from theformula shown in Suppary Fiur 1a.

For the temperature shift assay based on the population thermotaxis assay5,

wild-type worms, hsf-1 mutants, hsf-1-overexpressing worms and other mutants

were cultivated with food. On the first day, two wild-type worms or three mutants

were each deposited on the NGM plate and cultivated at 17 C. After 12 h , the

deposited postnatal day 0 (P0) worms were removed from each plate to segregate

F1 progeny. In the case of the temperature shift assay from 17 C to 23 C, F1

worms were shifted to a new temperature of 23 C after cultivation at 17 C for

4.5 d (after deposition at P0 on the NGM plate), and the population thermotaxis

assay was performed at each time point (Fi. 2a). In the case of the temperature

shift assay from 23 C to 17 C, F1 worms were initially cultivated at 17 C until

they passed the L1L2 larval stage and were then cultivated at 23 C because of

the L1L2 larval arrest phenotype inhsf-1 mutants at 25 C (ref. 19). After worms

grew to the adult stage, the temperature was shifted from 23 C to 17 C, and the

thermotaxis assays were conducted at each time point (Fi. 2b).To evaluate the effect of estrogen on the thermotactic behavior, 17-estradiol

(Sigma, E2275-1G) was applied to wild-type worms or several mutants. A 1,000-

fold concentrated solution of estradiol was prepared in dimethyl sulfoxide, and

the solution was added to each 6-cm plate containing 14 ml of agar and a bacterial

lawn to obtain the NGM plate containing estradiol at the final concentration.

Worms were cultivated on the NGM plate supplied with estradiol at 17 C or 23 C

during behavioral conditioning or until the adult stage; worms cultivated without

estradiol were cultivated with dimethyl sulfoxide as a vehicle control.

Quaiaiv PcR aaysis. Wild-type worms and hsf-1 mutants were individu-

ally synchronized as described above. In the case of the temperature shift from

15 C to 25 C, after cultivation at 15 C for 5 d, mRNA was isolated by RiboPure

kit (Ambion) just before shifting the cultivation temperature to a new temperature
http://discover.nci.nih.gov/gominer/http://discover.nci.nih.gov/gominer/


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nature neurOSCIenCe doi:10 1038/nn 2854

25 C and at 5 h after shifting temperature. In the case of the temperature shift

from 25 C to 15 C, both the wild-type worms andhsf-1 mutants were initially

cultivated at 17 C until they passed the L1L2 larval stage and then cultivated

at 25 C until the adult stage. The isolated mRNA (300 ng) was transcribed

and amplified using primers specific to the genes, SuperScript III Platinum

SYBR Green One-Step qRT-PCR Kit (Invitrogen) and a DNA Engine Peltier

Thermal Cycler-200 (Bio-Rad). To estimate the fold-change value (Fi. 3b and

Suppary Fis. 3 and 8i,j), the mRNA abundance of each gene isolated

from worms at 5 h after shifting the temperature from 15 C to 25 C or from25 C to 15 C was divided by that of the worms measured before shifting the

temperature. Expression changes of each gene were normalized to that of the

house-keeping gene lmn-1 as a reference.

caiu iai aaysis.In vivo calcium imaging was performed according to

previously reported methods9,28, but with some modifications. To monitor the

temperature-evoked response of each neuron, yellow cameleon 3.6 was expressed

in each worm. These worms were glued onto a 1.5% agar pad on glass, immersed

in M9 buffer and covered with a glass coverslip. The agar pad and M9 buffer were

kept at the initial imaging temperature. Sample preparation was completed within

2 min. The sample was then placed onto a Peltier-based thermocontroller (Tokai

Hit, Japan) on the stage of an Olympus BX61WI microscope at the initial imag-

ing temperature for 2 min, and fluorescence was introduced into a Dual-View

optics system (Molecular Devices). Cyan fluorescent protein (CFP; F480) and

yellow fluorescent protein (YFP; F535) images were simultaneously capturedby an electron-multiplying charge-coupled device camera, C9100-13 ImagEM

(Hamamatsu Photonics). For calcium imaging during transient up-and-down

warming and step-wise warming, images were taken with a 500-ms exposure

and 1 1 binning. For Fiur 8, to protect the probe from photobleaching

during measurement, each image was taken with a 100-ms excitation pulse at a

1-Hz frame rate using a pulse-generator-equipped imaging apparatus (SG-4115,

Iwatsu). The temperature on the agar pad was monitored by a thermometer sys-

tem, DCM-20 (Tokai Hit and Hamamatsu Photonics). Fluorescence intensities

of F535 and F480 were measured using the MetaMorph imaging analysis system

(Molecular Devices).

Saisia aaysis. The statistical analysis forFiurs 4i and6 was performed

using two-way analysis of variance followed bypost hoc analysis using the Fishers

protected least significant difference test, whereas that forFiurs 2, 3b,

5, 7a and 8a and Suppary Fiur 3 was conducted using t-tests.

Differences were considered to be significant when P< 0.05.

37. Mello, C.C., Kramer, J.M., Stinchcomb, D. & Ambros, V. Ecient gene transer in

C.elegans: extrachromosomal maintenance and integration o transorming

sequences. EMBO J.10, 39593970 (1991).

38. Von Stetina, S.E. et al. Cell-specic microarray proling experiments reveal a

comprehensive picture o gene expression in the C. elegans nervous system.

Genome Biol.8, R135 (2007).

39. Watson, J.D. et al. Complementary RNA amplication methods enhance microarray

identication o transcripts expressed in the C. elegans nervous system.

BMC Genomics9, 8497 (2008).

40. Irizarry, R.A. et al. Summaries o Aymetrix GeneChip probe level data. Nucleic

Acids Res.31, e15 (2003).

41. Murphy, C.T. et al. Genes that act downstream o DAF-16 to infuence the liespan

o Caenorhabditis elegans. Nature424, 277283 (2003).

Regulation of Behavioral Plasticity by Systemic Temperature Signaling in Caenorhabditis Elegans

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