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Regulation of behavioral plasticity by systemic temperature 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 between systemic environmental recognition and behavioral plasticity in the nervous system. Temperature is an unavoidable somatic stimulus that ultimately affects survival and reproduction. The impact of temperature has been clearly demonstrated by studies showing that change of core body temperature by means of changing the temperature of the hypothalamus alters the lifespan of transgenic mice 1. To confront environmental temperature change, animals must evolve a behavioral strategy 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 bodies2. Various poikilotherms have acquired the ability to memorize comfortable temperatures and to move to places that minimize tem perature deviation from the memorized temperatures. One such successful strategy of poikilotherms is observable in thermotaxis in the nematode C. elegans3,4: most worms that are cul tivated at a particular temperature between 15 C and 25 C for a few hours with a food source migrate to the cultivation temperature when placed on a thermal gradient for an hour5,6. In addition, worms that are conditioned to migrate to a certain temperature are capable of migrating to a new cultivation temperature a few hours after shifting to 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. Genomewide profiling upon behavioral conditioning indicated that perception of a change in normal cultivation temperatures by heatshock transcrip tion factor HSF1, 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 between approximately 15 C and 25 C), drives the expression dynamics of
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direct or indirect downstream genes not found among the classical heatshock response hsp genes. Expressions of HSF1 in body wall muscles or intestine restored the thermotactic defect of hsf-1 mutants, suggesting that nonneuronal cells can induce HSF1dependent downstream signaling in accordance with cultivation temperature changes. Genetic epistasis analysis and calcium imaging showed that this systemic signaling regulates noncell autonomously the thermo sensory neurons through the estrogen signaling pathway, leading to behavioral change. RESULTS Genome-wide profiling highlights HSF-1 To gain detailed molecular insight into thermotactic behavior, we performed a genomewide microarray analysis during behavioral conditioning 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 (Fig. 1ad and Supplementary Figs. 1 and 2). We detected 79 candidate genes, which are likely to include genes that are involved in thermotactic behavior in addition to genes that are differentially expressed simply as a result of temperature shift. Examining the candidate genes, we noticed sub stantial changes in expression of several hsp genes, which are regulated by HSF1 (ref. 10). HSF1 is highly conserved from nematodes to mam mals11. Previous structural and functional characterizations of HSF1 revealed that the transient heatshock stimuli enable HSF1 to change structurally from an autoinhibited monomer to a trimer and to trans locate to the nucleus, thereby activating gene transcription10,1216. Thus, HSF1 is well known to directly perceive transient heatshock stimuli. Notably, previous in vitro studies have shown that normal growth tem peratures, as well as transient heatshock stimuli, can induce the reversi ble structural transition of HSF1 and its DNA binding17,18. Considering these previous observations and our own (Supplementary Fig. 2),

of Molecular Neurobiology, Graduate School of Science, Nagoya University, Nagoya, Japan. 2CREST, Japan Science and Technology Agency, Tokyo, Japan. for Advanced Research, Nagoya University, Nagoya, Japan. 4Present address: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto, Japan. Correspondence should be addressed to I.M. (m46920a@nucc.cc.nagoya-u.ac.jp). Received 1 April; accepted 3 May; published online 26 June 2011; doi:10.1038/nn.2854

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Figure 1 The essential role of HSF-1 in a C er C Cultivated aft 7 17 memory-based behavior revealed by genomeat 23 C 4 h to 1 t to g hif h n S 0 ifti wide microarray analyses. (a) Procedure for sh at microarray analysis of the thermotactic (TTX) behavior conditioning process. C. elegans Linear TTX assay Linear TTX assay Microarray Microarray were cultivated at 23 C for 2.5 d and were 4.00 then shifted to a new temperature of 17 C. 3.00 2.00 Immediately after start of the assay, we isolated 1.00 mRNA from worms that were not used for the 0 1 2 3 4 5 6 behavioral assays. Error bars represent s.e.m. 1.00 0 2.00 Time (h) (bd) Overall transition of gene expression 3.00 change during behavioral conditioning. 4.00 Scatter plot of intensity values (log2) for a 14 14 14 representative hybridization of RNA isolated 12 12 12 from worms at 1 h (b), 2 h (c) or 4 h (d) after 10 10 10 shifting the cultivation temperature from 8 8 8 23 C to 17 C compared with that isolated 6 6 6 from worms just before shifting the cultivation 4 4 4 temperature. A.u., arbitrary units. (ei) The 2 2 2 HSF-1 activity within a range of cultivation 2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14 temperatures in vivo. The hsp-16.2p::gfp Intensity at 0 h (a.u.) Intensity at 0 h (a.u.) Intensity at 0 h (a.u.) reporter gene, including the two HSF-1 binding 40 m 40 m 40 m elements (HSEs), was expressed in a wild-type background (e). Each worm was cultivated hsp-16.2p with food at 15 C for 7 d. The GFP (500 bp) observations were performed before shifting gfp the temperature (f) and at 2 h after shifting Cultivation at 15 C 2 h after shifting to 23 C 2 h after shifting to 25 C HSF-1 binding element to the new temperatures (which were within 40 m the range of normal cultivation temperatures); 23 C (g) or 25 C (h). For the transient heatshock treatment (i), each worm was incubated at 35 C for 15 min and then allowed to recover at 15 C for 2 h before the GFP observation. The GFP observations were conducted at least four times in each experimental condition.

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we wished to address the new biological importance of the role of HSF1 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 and 25 C) activates C. elegans HSF1 by observing the green fluorescent protein (GFP) fluorescence of wildtype strains expressing hsp-16.2 promoter::gfp reporter, whose promoter contains two heatshock tran scription factor binding elements (HSEs) (Fig. 1e). We observed large GFP fluorescence changes that were dependent on two HSEs at 2 h after shifting the temperature from 15 C to 23 C or to 25 C (Fig. 1fh and Supplementary Fig. 3ak), which were comparable with those observed at 2 h after the transient heatshock treatment (Fig. 1i). As HSF1 activity responded to ambient cultivation temperatures, we addressed whether HSF1 is required for thermotactic behavior. hsf-1(sy441) mutants19, carrying a reductionoffunction mutation (see Supplementary Strains and plasmids), migrated to a cultiva tion temperature to which they had previously been habituated when cultivated at 17 C (Supplementary Fig. 4a,b,d), whereas mutants cultivated at 23 C did not migrate to the cultivation temperature (Supplementary Fig. 4a,c,e). 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) and instead migrated to colder temperatures (Fig. 2a). At 5 h after shift ing the cultivation temperature from 23 C to 17 C, they showed a tendency to migrate toward the new cultivation temperature of 17 C (Fig. 2b). Worms carrying the hsf-1(ok600) null mutation as either a homozygote or heterozygote showed a behavioral defect similar to that of the hsf-1(sy441) mutants (Fig. 2c). We next addressed whether the abnormal thermotactic migration of hsf-1(sy441) mutants is merely a reflection of slower temperature
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memory acquisition: might they eventually become conditioned to 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 C to 23 C (Fig. 2d). Excess expression of native HSF1 in wildtype worms10 also induced partially abnormal migration, which was partly reminiscent of the defect of hsf-1(sy441) mutants (Fig. 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 also showed a strong tendency to migrate toward colder regions than hsf-1 overexpression lines, although that difference was not statistically significant (Fig. 2d). 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 HSF1 is important for proper thermotactic response. The thermotaxis defect in hsf-1(sy441) mutants (hereafter referred to as hsf-1 mutants) might simply reflect their temperaturesensitive debility19, thus causing limited migration ability for all sensory cues at 23 C. We therefore addressed whether other sensory responses, such as odorant chemotaxis20,21 of hsf-1 mutants cultivated at 23 C, are more defective than those cultivated at 17 C (Supplementary Fig. 3mo). The hsf-1 mutants cultivated at 23 C did not exhibit olfactory defects of greater severity than the 17 Ccultivated mutants. Moreover, hsf-1 mutants cultivated at 23 C moved freely like wild type worms on the thermotaxis plate without a thermal gradient
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Figure 2 Characterization of thermotaxis Wild type 3.00 3.00 hsf-1(sy441) mutant behavior of hsf-1 mutants. (a,b) Population 2.00 2.00 hsf-1 overexpression thermotaxis (TTX) temperature shift assay Time (h) of wild-type worms, hsf-1(sy441) mutants 19 1.00 1.00 1 2 3 4 5 and hsf-1-overexpressing worms 10. 0 0 3 4 5 2 Cultivation temperature was shifted from 1 Time (h) 1.00 1.00 17 C to 23 C (a) and vice versa (b). The Wild type population TTX assay was conducted just 2.00 2.00 hsf-1(sy441) mutant before shifting the cultivation temperature hsf-1 overexpression 3.00 3.00 (0 h) and at every 1 h after shifting the temperature. (c) Thermotaxis of 4.00 4.00 * ** hsf-1(ok600) null mutants. Population ** TTX assays were conducted at 2 h after ** ** ** 3.00 3.00 3.00 NS NS shifting the temperature from 17 C to NS ** 23 C. **P < 0.01 (t-tests, n = 3). 2.00 2.00 2.00 (d) Evaluation of the effect of longterm cultivation at 23 C after shifting 1.00 1.00 1.00 temperature, on the thermotaxis of hsf-1 mutants and hsf-1-overexpressing worms. 0 Population TTX assays were conducted at 0 0 7 h and 10 h after shifting temperature from 17 C to 23 C. *P < 0.05, 1.00 1.00 1.00 **P < 0.01 (t-tests, n = 3). (e) Assessment of the dominant negative effect of HSF-1 on 7 h after shifting 10 h after shifting thermotaxis in the adult stage of wild-type to 23 C to 23 C worms. HSF-1DN was expressed in wild-type worms under hsp-16.2 promoter (hsp-16.2p::hsf-1dn). Population TTX assays were performed at 3 h after heat-shock treatment followed by the temperature shift from 17 C to 23 C. **P < 0.01 (t-tests, n = 3). NS, not significant. Error bars represent s.e.m.
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(Supplementary Fig. 5bf). These results suggest that the abnormal thermotactic behavior of hsf-1 mutants does not simply arise from temperaturesensitive debility but instead reflects a defect in the mechanism specific for thermotaxis. To examine whether HSF1 acts after a temperature shift from 17 C to 23 C, we constructed worms expressing in a wildtype background an HSF1 dominant negative form (HSF1DN)22 under the hsp-16.2 promoter and induced expression of HSF1DN by heatshock treat ment after worms had reached the adult stage. As the activity of the hsp-16.2 promoter itself is regulated by HSF1, we coinjected hsp16.2p::gfp to confirm whether expression of HSF1DN appropriately inhibits the endogenous HSF1 activity by monitoring the GFP fluo rescence from this reporter gene. Just before heatshock treatment, we observed hardly any fluorescence from worms carrying only the reporter gene and from those carrying hsp-16.2p::hsf-1dn and the reporter gene, and showed that both groups appropriately migrated toward lower temperature regions of around 17 C on the thermal gradient (data not shown). The wildtype worms carrying only the reporter gene and those carrying both hsp-16.2p::hsf-1dn and the reporter gene were then subjected to the transient heatshock 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, wildtype worms carrying both hsp-16.2::hsf-1dn and the reporter gene showed slightly less fluorescence than wildtype worms carrying only the reporter gene (Supplementary Fig. 6), sug gesting that HSF1DN inhibited the activity of endogenous HSF1. Moreover, wildtype worms carrying both hsp-16.2::hsf-1dn and the reporter gene showed abnormal cryophilic behavior compared with wildtype worms without both hsp16.2::hsf-1dn and the reporter gene (data not shown) and compared to those carrying only the reporter gene (Fig. 2e). These results suggest that HSF1 functions after shift ing the temperature from 17 C to 23 C. The thermotactic defects characteristic of hsf-1 mutants suggest that HSF1 is required for responding to higher temperature, for memorizing higher tempera ture or for both processes.
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Importance of HSF-1 downstream signaling in thermotaxis Notably, thermotactic behavior was barely affected by the tran sient heatshock treatment, although as expected markedly affected by shifting the cultivation temperature (Fig. 3a). To identify the genes involved in the HSF1 downstream signaling that is involved in regulating thermotaxis, we classified the genes that were dif ferentially expressed under microarray analyses into functional categories (Supplementary Fig. 1b) and then explored whether expression changes of representative genes in each category are HSF1dependent. After shifting the temperature from 15 C to 25 C, or vice versa, amounts of mRNA for hsp genes (Supplementary Fig. 3l) were substantially changed in wildtype worms, whereas changes in mRNA amounts were smaller in hsf-1 mutants. Similarly, the HSF1dependent expression changes were observed for genes encoding proteins that potentially have catalytic activity, such as DHS4 (hydroxysteroid 17 dehydrogenase 11) (Fig. 3b); genes encoding ion and peptide transporters, such as PGP9 (ABC trans porter superfamily) (Fig. 3c); and other gene functions (Fig. 3d). Thus, cultivation temperaturedependent changes in expression of genes with certain functions seem to occur in an HSF1dependent manner. Consistent with the importance of HSF1 in thermotaxis, mutants for kgb-2 (Jun Nterminal kinase), dhs-4, kqt-2 (KvQLT family potassium channel), pgp-9 and max-1 (a regulator of axon guidance) showed abnormal thermotactic migration (Fig. 3e). By contrast, the amounts of mRNA for genes such as cgt-1 (ceramide glucosyltransferase), pmp-1 (longchain acylCoA transporter) and dmd-7 (the transcription factor doublesex) changed inde pendently of HSF1 (Fig. 3bd). Mutants for these genes showed almost normal thermotaxis (Fig. 3e). 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 HSF1, and that HSF1dependent changes in downstream signaling are likely to be important for thermotaxis.
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HSF-1 signaling and the circuit We investigated how HSF1mediated sig naling regulates the thermotactic neural cir 1.00 cuit4,8,9 (Fig. 5). In this circuit, the bilateral ** AFD neurons are the principal thermosensory ** * 2.00 * * neurons and the AWC neurons are secondary * ** 3.00 thermosensory neurons4,9,23. Three guanylate Wild type grown at 17 C Mutant grown at 17 C cyclases GCY23, GCY8 and GCY18and the Gprotein subunit ODR3 are responsible Behavioral regulation by systemic temperature perception for the primary temperature signaling in AFD and AWC thermosensory We next explored where HSF1 functions to regulate thermotactic neurons, respectively, and all of these guanylate cyclases are upstream of behavior (Fig. 4). The GFP fluorescence expressed under the con the cyclic GMPgated channel TAX4 (ref. 8). hsf-1;gcy-23 gcy-8 gcy-18 trol of the hsf-1 promoter was detected in nearly all cells (Fig. 4a). quadruple mutants and hsf-1;odr-3 mutants cultivated at 23 C both Substantial fluorescence was detected in body wall muscles and intes showed a roughly intermediate thermotactic phenotype between that of tine (Fig. 4c,d), whereas relatively dim expression was observed in hsf-1 mutants and that of gcy-23 gcy-8 gcy18 or odr-3 mutants, respec neurons (Fig. 4b). tively (Fig. 5a,b,d). It was difficult to analyze the genetic epistasis between To explore the cells in which the HSF1 activity is required for hsf-1 and tax-4 owing to the overlapping phenotypes of the single mutants thermotaxis, we expressed hsf-1 cDNA in hsf-1(sy441) mutants (data not shown). Expression of hsf-1 in the body wall muscles of the using several promoters (Supplementary Fig. 5a) to drive HSF1 quadruple mutants (hsf-1;gcy-23 gcy-8 gcy-18; Ex[myo-3p::hsf-1]) resulted expression in various sets of cells. Expression of hsf-1 cDNA in in a defect almost identical to that of gcy-23 gcy-8 gcy-18 triple mutants, nearly all neurons, or coordinately in AFD, AWC, AIZ and RIA suggesting that hsf-1 expression from the body wall muscles restored neurons, which comprise the thermotactic circuit, was sufficient the behavioral defect caused by the hsf-1 mutation in hsf-1;gcy-23 gcy-8 to restore the normal thermotactic migration of hsf-1 mutants gcy-18 quadruple mutants (Fig. 5a,d and Supplementary Fig. 6a). Previous studies have indicated that ttx-3 encodes LIM homeo (Fig. 4e,g), and expression of hsf-1 cDNA in several indivi dual neurons of the neural circuit weakly restored the behavior domain protein and that ttx-3 mutants show a thermotaxis (TTX) (Fig. 4h,i). Notably, consistent with the expression pattern of HSF1 defect that mimics the defect seen upon ablation of AIY inter (Fig. 4c,d), expression of hsf-1 cDNA in body wall muscles or in neurons24, which are postsynaptic to AFD and AWC neurons. The intestine almost fully rescued the defect of hsf-1 mutants (Fig. 4f). main effect of the ttx-3 mutation is known to be disruption of the Thus, nonneuronal expression of HSF1 is sufficient for the resto projection pattern of the AIY neurites2426. Thus, the thermotactic ration of thermotaxis. Together, these results led us to hypothesize defect caused by ttx-3 mutation probably results from the functional that HSF1 acts throughout the body and that nonneuronal and role of TTX3 in AIY. We therefore analyzed genetic epistasis between neuronal cells respond to environmental temperatures to convey hsf-1 and ttx-3. hsf-1;ttx-3 mutants showed a behavioral defect very noncellautonomous signals to the thermotactic neural circuit that similar to that of ttx-3 single mutants (Fig. 5c,d), suggesting that ttx-3 generate behavioral output. was genetically epistatic to the HSF1 signaling. Altogether, our
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Figure 3 Thermotaxis controlled by HSF-1 downstream signaling. (a) Behavioral effects of cultivation temperature shift and heat-shock treatment, both of which activate HSF-1. The thermotaxis (TTX) assays were also performed at 2 h after shifting the temperature from 17 C to 23 C or at 2 h during recovery after the transient heat-shock treatment at 34 C for 15 min (n = 3). (bd) Identification of HSF-1 downstream genes. Expressions of genes possessing the catalytic activity (b), genes encoding ion and peptide transporter (c) and other functional genes (d) were analyzed using quantitative PCR in wild-type worms and hsf-1 mutants. mRNAs were isolated from each worm before and at 5 h after shifting the cultivation temperature from 15 C to 25 C or from 25 C to 15 C. Genes that possess HSEs within their 6.0-kb promoter regions are underlined. *P < 0.05, **P < 0.01 (t-tests, n 3) in a comparison between wild-type worms and hsf-1 mutants. (e) Thermotaxis of worms carrying mutations in putative downstream genes of HSF-1. The importance of each gene in thermotaxis was investigated using available mutants. Worms were cultivated at 23 C for 2.5 d or 17 C for 4.5 d. *P < 0.05, **P < 0.01 (t-tests, n = 3), in a comparison of each mutant with wild-type worms. Error bars represent s.e.m.

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Figure 4 Cell-specific rescue of defective thermotactic behavior in hsf-1 mutants. (ad) Expression pattern of HSF-1 in wild-type worms. To drive the GFP fluorescence under the hsf-1 promoter, hsf-1 promoter::gfp reporter gene was injected into the wild-type worms. The GFP fluorescence was detected in nearly all cells (a): for example, in the nerve ring (b), body wall muscles (c) and intestine (d). (ei) Cell-specific rescue experiments. Thermotactic behavior was examined at 2 h after shifting temperature from 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) of hsf-1 mutants using individual cell-specific 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 least two lines were assayed for each transgenic strain. Experiments for each transgenic strain were carried out at least three times. The significant differences of thermotactic plasticity between each transgenic strain and hsf-1(sy441) mutants were determined by Fishers protected least significant difference multiple-comparison test. *P < 0.05, **P < 0.01. Error bars represent s.e.m.

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genetic epistasis analysis suggests that the HSF1mediated non cellautonomous 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 (Supplementary Fig. 7). Although recent studies addressed whether the HSF1 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 heatshock stimuli and cultivation temperature changes were not substantially different between wildtype worms and gcy-23 gcy-8 gcy18 triple mutants, or ttx-3 mutants (Supplementary Fig. 8). We also showed that the GFP fluorescence level of nonneuronal cells, such as intestine in gcy-23 gcy-8 gcy-18 mutants or ttx-3 mutants expressing hsp-16.2 reporter constructs, is comparable to the fluorescence of those in wildtype worms. The results in this study were consistent with a recently reported result suggesting that the HSF1 activity is independent of the ttx-1 mutation that is known to specifically disrupt AFD structure27, but partly contradicted a previous study16 that found the AFD and AIY neurons to be important for the expression of a few hsp genes. This contradiction might arise from the subtle difference in growth conditions. In particular, humidity has been known to affect temperature sensation and thermotaxis, although little is known about the mechanism dictating the relationship between hygrosensation and thermosensation. Taken together, at least in our growth condition, expression of a few HSF1 downstream genes such as hsp-70 seems to be independent of gcy-23 gcy-8 gcy-18 and ttx-3 mutations. Regulation of the thermosensory neurons by HSF-1 signaling To address the possibility that the HSF1 signaling affects AFD and AWC sensory neurons, we monitored temperature stimulusevoked
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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-1 mutants than in wildtype worms (Fig. 6a). By contrast, AWC in hsf-1 mutants was more responsive to the transient temperature change from 17 C to 23 C than AWC in wildtype worms (Fig. 6b). These results suggest that the HSF1 signaling is involved in modulation of the 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 mutants were substantially less than those in wildtype worms, probably owing to the decreased and increased responsiveness of AFD and AWC to transient temperature changes in hsf-1 mutants, respectively (Supplementary Fig. 9). Expression of hsf-1 in body wall muscles restores the temperature responsiveness of AFD, AWC and AIY in hsf-1 mutants (Fig. 6a,b and Supplementary Fig. 9b). These results are consistent with the results from genetic epistasis analysis, which found that the HSF1 signaling regulated AFD and AWC (Fig. 5). Previous calcium imaging experiments have shown that, when tem perature is raised from 15 C to 25 C in a stepwise manner, AFD and AWC neurons respond to every warming above a threshold tem perature that is set by the previous cultivation temperature, indicating
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Figure 5 Genetic interactions between HSF-1 signaling 4.00 ** and the genes that act in the thermotactic neural circuit. ** ** NS 3.00 (ac) Genetic epistasis analysis. Thermotactic plasticity hsf-1; of wild-type worms and each mutant was examined using gcy-23 2.00 hsf-1; gcy-8 population thermotaxis (TTX) assays at 2 h after shifting gcy-23 gcy-23 gcy-18 1.00 gcy-8 Ex[myo-3p:: gcy-8 the temperature from 17 C to 23 C. The TTX plate gcy-18 gcy-18 hsf-1] ttx-3 hsf-1;ttx-3 was divided into eight regions, 4 (coldest) to +4 0 hsf-1; odr-3 Wild type hsf-1 (warmest) (Supplementary Fig. 1a). Epistatic relations odr-3 1.00 between hsf-1 mutants and gcy-23 gcy-8 gcy-18 mutants (a), odr-3 mutants (b) or ttx-3 mutants (c) are 2.00 NS shown. The ttx-7;hsf-1 double mutants were also 3.00 constructed, but it was difficult to analyze their ** thermotactic behaviors owing to their severe locomotory defects. (d) Summary of genetic epistasis analysis for the relationship between systemic temperature perception mechanism and thermotactic neural circuit. Thermotactic plasticity was examined as above. The significance of differences of thermotactic plasticity was determined using the Fishers protected least significant difference multiple-comparison test. **P < 0.01; NS, not significant. Error bars represent s.e.m.
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that these thermosensory neurons can, of themselves, memorize the perceived cultivation temperature9,28. To investigate the responsive temperature range, we also measured the FRET ratio changes of AFD neurons of wildtype worms and hsf-1 mutants during the stepwise warming (Fig. 6). Consistent with the results from the previous cal cium imaging experiments28, the AFD neuron of wildtype worms cultivated at 23 C showed a marked response when warmed to a temperature above 23 C (Fig. 6c). By contrast, the AFD neuron of hsf-1 mutants cultivated at 23 C showed a marked response when warmed to a temperature above 21 C; that is, the response of hsf-1 mutants to warming occurred earlier than that of wildtype worms (Fig. 6c,e). There was, however, hardly any observable difference in the cultivationtemperature dependency of AWC response between wildtype worms and hsf-1 mutants (Fig. 6d). Furthermore, expres sion of hsf-1 in body wall muscles sufficiently rescued an abnormal response of the AFD neuron of hsf-1 mutants (Fig. 6c,e). These results indicate that the HSF1 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 HSF1 signaling regulates the thermo sensory neurons. Among genes likely to act downstream of HSF1, dhs-4 and cyp-37B1 genes encode hydroxysteroid 17 dehydroge nase 11 (Wormbase gene ID, WBGene00000968) and a cytochrome P450 CYP4/CYP19/CYP26 subfamily protein (WBGene00009226), respectively, both of which are believed to be involved in estrogen synthesis. In a local alignment search using the sequences of Homo sapiens estradiol 17 dehydrogenase 1 (HSD17B1) and Homo sapiens cytochrome P450 19A1, known as aromatase (CYP19A1) as queries against the C. elegans genome database, both DHS4 (25% identity, expected value (Evalue) = 2 1021) and CYP37B1 (23% identity, Evalue = 3 109) were ranked in the top five matches. Recent reports have shown that estrogen and its receptor are involved in hippocampal synaptic plasticity and memory through the regulation of calcium influx2931. We therefore assessed the effect of estrogen signaling on C. elegans thermotaxis.
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We exogenously applied 17estradiol (referred to as estradiol from here on), a main form of estrogen, to wildtype worms grown at 23 C from their birth. An application of estradiol caused thermophilic movement in a dosedependent manner (Fig. 7a). Further, estradiol application on 23 Ccultivated hsf-1 mutants and dhs-4 mutants par tially restored their behavioral defects (Fig. 7b,c). Notably, estradiol application only during the behavioral conditioning process caused thermophilic movement of wildtype worms and partially rescued the behavioral defect of hsf-1 mutants and dhs-4 mutants (Fig. 7d and Supplementary Fig. 1c). Despite the clearly observable effects of estradiol on wildtype worms as well as hsf-1 mutants and dhs-4 mutants, estradiol application to ttx-3 mutants did not induce any obvious effect, suggesting that ttx-3 is genetically epistatic to estrogen signaling as well as to the HSF1 signaling (Fig. 5c,d and 7bd). On the basis of the results from the estradiol application experi ments, we investigated the relationship between HSF1 signaling and estrogen signaling. The previous in vitro binding analysis showed that the three nuclear receptors NHR14, NHR69 and NHR121, all of which were identified by their highest homologies to human estrogen receptor, bound estradiol in a dosedependent fashion32. We analyzed thermotaxis of mutants impaired in the nhr-14, nhr-69 or nhr-121 genes. nhr-69 mutants cultivated at 23 C showed a sub stantial tendency to migrate toward colder regions than wildtype worms, whereas nhr-14 mutants and nhr-121 mutants cultivated at 17 C or 23 C showed no obvious defect (Fig. 8a). We further inves tigated the functional redundancy between NHR69 and NHR14 or NHR121, examining thermotaxis of nhr-69;nhr-121 mutants, nhr69;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-14 mutants were each similar to that of nhr-69 single mutants (Fig. 8a), suggesting that there is no redundancy between nhr-69 and the other two nhr genes under the 23 Ccultivated condition. By contrast, nhr-69;nhr-121 mutants, nhr-69;nhr-14 mutants and nhr-69;nhr-121;nhr-14 mutants each cultivated at 17 C exhibited a slightly more severe behavioral defect than 17 Ccultivated nhr-69 mutants (Fig. 8a), suggesting that
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NHR69 functions redundantly with NHR14 and NHR121 under the 17 Ccultivated condition. Taken together, these data suggest that at least NHR69 is required for thermotaxis. We then conducted a genetic epistasis analysis between nhr-69 and hsf-1. The behavior of nhr-69 hsf-1 double mutants was similar to that of nhr-69 mutants (Fig. 8b), indicating that nhr-69 is epistatic to hsf-1. This result suggests that NHR69, which might mediate both thermophilic and cryophilic movements, could act downstream of the HSF1 signaling to be involved in the former movement. To further address this possibility, we took a pharmacological approach to test whether estradiol application affects thermotaxis of nhr-69 mutants. Estradiol application had no substantial effect on the thermotactic behavior of nhr-69 mutants (Fig. 8c,d). Considering the effect of estra diol application on hsf-1 mutants (Fig. 7bd), this result is consistent with the idea that NHR69 acts downstream of the HSF1 signaling.

Given that ttx-3 is epistatic to estrogen signaling (Fig. 7bd), we further attempted to identify a site of action of NHR69. We examined whether the thermotactic behavior of nhr-69 hsf-1 double mutants was restored to that of hsf-1 single mutants when NHR69 is expressed in AFD, AWC or AIY neurons of nhr-69 hsf-1 mutants. The expression of nhr-69 cDNA in AWC or AIY neurons had no obvious effects on the behavior of nhr-69 hsf-1 mutants, but strong expression of nhr-69 (3 ng l1) in the AFD neuron of nhr-69 hsf-1 mutants caused a strong tendency to migrate toward colder regions rather than a restoration to the phenotype of hsf-1 single mutants (Fig. 8b). These results suggest that NHR69 acts in AFD, although excess expression of nhr-69 in AFD on the HSF1 signalingreduced background leads to an increase in inactive NHR69 in the AFD, which might cause a stronger tendency for nhr-69 hsf-1 mutants to migrate to colder regions. Indeed, weaker expression of nhr-69
Figure 7 Effect of estradiol application on thermotaxis. (a) The dosedependent effect of estradiol application. Wild-type worms were cultivated at 23 C with indicated concentration of estradiol. To clearly observe the thermophilic movement of worms cultivated at 23 C, the center of the thermotaxis (TTX) plate was adjusted to 23 C to establish a linear thermal gradient ranging from approximately 20 C to 26 C. However, other behavioral assays were conducted by adjusting the center of the TTX plate to 20 C to establish a thermal gradient ranging from approximately 17 C to 23 C (see Supplementary Fig. 1). (b,c) Effect of exogenously applied estrogen on thermotactic behaviors of wild-type worms and several mutants. Each worm was cultivated at 23 C (b) or 17 C (c), either with (on) or without (off) 10 M estradiol. (d) Effect of 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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0 30 60 Figure 6 Regulation of the thermosensory neurons by the HSF-1 signaling. (a,b) In vivo calcium imaging of 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 of warming was used to examine calcium transients in AFD (a) and AWC (b). The average ratio changes of AFD and AWC were measured for wild-type worms (blue), hsf-1 mutants (red) and hsf-1 mutants expressing hsf-1 in body wall muscles (green). Stimulus temperatures are shown at the bottom. n = 11 to 17 worms. (ce) In vivo calcium imaging of 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 of 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 for wild-type worms (blue), hsf-1 mutants (red) and hsf-1 mutants expressing hsf-1 in body wall muscles (green). Arrows indicate the first marked ratio changes during stepwise warming. The average ratio change of the AFD neurons in response to the temperature change from 19 C to 21 C during step-wise warming is shown in a bar graph (e). **P < 0.01; NS, not significant (t-tests, n = 12 to 20 worms). Error bars represent s.e.m.

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Figure 8 HSF-1 signaling acts through estrogen signaling to regulate the AFD thermosensory neurons. (a) The thermotactic (TTX) behavior of 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 of worms was examined 2 h after shifting temperature from 17 C to 23 C. Concentrations of nhr-69 cDNA expressed in nhr-69 hsf-1 mutants under cellspecific promoter were indicated. (c,d) Effect of estradiol on thermotaxis of nhr-69 mutants. Worms were grown at 23 C (c) or 17 C (d) with or without 100 M of estradiol. (e) Calcium imaging of 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-69 in AFD neurons at the concentration of 0.3 ng l1 (n = 15) or 3 ng l1 (n = 13). **P < 0.01; NS, not significant (t-tests). Error bars represent s.e.m.

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2011 Nature America, Inc. All rights reserved.

Wild type nhr-69 hsf-1 nhr-69 hsf-1 worms expressing 1 gcy-8p::nhr-69 (0.3 ng l ) nhr-69 hsf-1 worms expressing 1 gcy-8p::nhr-69 (3 ng l )

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(0.3 ng l1) in AFD restored the thermo tactic behavior of nhr-69 hsf-1 double mutants Wild type nhr-69 10 d (Fig. 8b). To further examine whether the Off On Off On 0 expression of nhr-69 in AFD affects the AFD 0 1.00 activity in 23 Ccultivated nhr-69 hsf-1 23 C mutants, we measured calcium transients 2.00 17 C 17 C of the AFD in nhr-69 hsf-1 mutants and the NS 3.00 NS mutants expressing nhr-69 cDNA in the AFD 0 30 60 90 120 150 180 210 240 Time (s) at 0.3 ng l1 or 3 ng l1. Consistent with the results from behavioral assays (Fig. 8b), expression of nhr-69 in the AFD neuron of nhr-69 hsf-1 mutants gradient in population assays (Fig. 5ac). It is probable that C. elegans affected the AFD activity in a dosedependent manner (Fig. 8e). possesses multiple types of thermosensory systems that are not mutu Together with the results from the exogenous estrogen application, ally exclusive. Given the recent studies indicating that the sensory system genetic and calcium imaging analyses, this suggests that HSF1 sig naling acts at least in part through estrogen signaling, directly or operating behavior is more diverse than previously thought 33, it is reasonable to consider the possibility that animals can perceive envi indirectly, to regulate the thermosensory neuron AFD. ronmental changes not only through a specialized sensory system DISCUSSION but also through sensors distributed across the body. For example, a In the current study, we found that systemic temperature signal small set of warmthactivated anterior cell neurons is located in the ing regulates the thermosensory neurons for thermotaxis through internal portion of the Drosophila melanogaster brain, whose func heatshock transcription factor and estrogen signaling. The heat tion is important for preferred temperature selection 34. In addition, shock transcription factor has been well known as a thermosensor D. melanogaster larvae perceive light by using neurons embedded that is highly conserved from organisms as distant as Saccharomyces under the cuticle encasing their bodies35. This sensory system does cerevisiae. By contrast, the AFD neuron is a specialized thermosensor not eliminate the importance of photoreceptors in eyes. In C. elegans, with sensitivity enough to respond to differences of 0.01 C on a the PVD neurons that are located on either side of the worm and thermal gradient8. We therefore infer that C. elegans added its fine that envelop the body with multidendritic processes respond to cold temperature sensitivity through the evolution of the AFD thermosen temperatures36. Thus, it is possible to suppose that an HSF1mediated sory system. Given that the HSF1 protein responds to temperature mechanism might be responding to a signal from other neurons, through its multimerization ability, it is unlikely that HSF1 is as sen such as PVD neurons. Although further work is needed to clarify sitive as AFD. Alternatively, as a plausible purpose of the temperature the relationship among the various temperature signaling systems, response, HSF1 might function during behavioral conditioning after this study and others3336 suggest that sensory systems influencing a temperature shifts from 17 C to 23 C in an incubator, as microarray behavior might be broadly required throughout an animals body. A principal view in neuroscience is that environmental temperature analysis, quantitative PCR analysis and GFP observation indeed indi cated that expression of several genes downstream of HSF1 changed is detected by sensory neurons in the nervous system. We demon during this process (Figs. 1eh and 3bd and Supplementary Figs. 2 strate here that temperature signaling by nonneuronal cells triggers and 3a,b,l). Although the AFD neurons can respond to temperature behavioral changes, as thermosensory neurons do. Given the robust differences of 0.01 C for isothermal tracking, worms were broadly influence of temperature on animals bodies, it improves behavioral distributed around the cultivation temperature in the temperature performance to monitor environmental temperatures at the level of
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the entire body. To ensure that this sort of strategy is retained through evolution, heatshock factor, a highly conserved protein, might adopt a role in transcriptionally coupling environmental recognition to a memoryregulated 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/. Accession codes. 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 (Stanford University School of Medicine) for pPD plasmids; Y. Iino (University of Tokyo) for the gcy-5 and gcy-7 promoters; Caenorhabditis Genetic Center, C. elegans Knockout 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 this manuscript and discussion. I.M. is a Scholar of the Institute for Advanced Research in Nagoya University, Japan. This work was supported by the Core Research for Evolutional Science and Technology (CREST) Program of the Japan Science and Technology (JST) agency (to I.M.). AUtHoR contRIBUtIonS T.S. designed the research, performed most experiments, analyzed data and wrote the manuscript; Y.N. performed the quantitative PCR experiments and conducted germline transformation to construct the C. elegans transgenic line; I.M. supervised the project, conducted initial identification of cells expressing the hsf-1 promoter::gfp reporter 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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genetics. The hsf-1(sy441) mutation19 was outcrossed to the wild type six times. The sy441 mutant has a single GtoA mutation in the seventh exon (5AGT AAT CAT AAT TGG GAT GAT TTT GGG AAT3), 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. germline transformation. Germline transformations were performed as described37. For cellspecific rescue experiments, coinjection mixes consisted of pKDK66 ges-1p::NLS GFP (50 ng l1) and experimental DNA at various concentrations (1 or 10 ng l1). To analyze the expression pattern of HSF1, 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 coinjected with pNAS88 ges-1p::NLS TaqRFP (50 ng l1) and pTAN124 ges-1p::NLS DsRed monomer (50 ng l1), respectively. mRnA isolation and amplification. For synchronization of worm growth38, worms were initially grown to starvation in a 60mm 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 150mm plate. The resultant plates were incu bated at 23 C until most of the food was depleted. The worms were collected from 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 200ml 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 53m 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 150mm 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 Sbasal 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 shifting temperature to 17 C (1, 2, 3 or 4 h at 17 C) (Fig. 1a). Total RNA was isolated from the lysate using the RiboPure kit (Ambion). The highfidelity linear amplification of 40 ng of isolated mRNA was accom plished using the WTOvation Pico RNA Amplification System (NuGEN) according to the manufacturers protocol, with some modifications39. The ampli fied cDNA was fragmented and biotinylated using FLOvation cDNA Biotin Module V2 (NuGEN). microarray. Fragmented, biotinlabeled cDNA (50 l) 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 same array 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. Analysis of microarray data. A twoclass paired analysis of the data was per formed to identify genes that differed by 1.8fold 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 (Supplementary Fig. 2) were found to be significantly regulated, with 6.813 median false significant genes. The 79 differently expressed genes were

ONLINE METHODS

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 observation. All observations were carried out using an Axioplan2 light microscope (Carl Zeiss) except for those in Figure 4ad, 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 transferred to 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 Supplementary Figure 3k. The GFP intensity of each worm was calculated as described in the figure legend of Supplementary Figure 3 and was quantified using ImageJ software (US National Institutes of Health). thermotaxis assay. A radial temperature gradient assay was carried out using a 9cm 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, 60cm 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 ml of 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 13.5cmlong 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 6cm 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 Sbasal 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 the formula shown in Supplementary Figure 1a. For the temperature shift assay based on the population thermotaxis assay5, wildtype worms, hsf-1 mutants, hsf-1overexpressing worms and other mutants were cultivated with food. On the first day, two wildtype 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 (Fig. 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 in hsf-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 (Fig. 2b). To evaluate the effect of estrogen on the thermotactic behavior, 17estradiol (Sigma, E22751G) was applied to wildtype worms or several mutants. A 1,000 fold concentrated solution of estradiol was prepared in dimethyl sulfoxide, and the solution was added to each 6cm 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. Quantitative PcR analysis. Wildtype 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

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doi:10.1038/nn.2854

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25 C and at 5 h after shifting temperature. In the case of the temperature shift from 25 C to 15 C, both the wildtype worms and hsf-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 OneStep qRTPCR Kit (Invitrogen) and a DNA Engine Peltier Thermal Cycler200 (BioRad). To estimate the foldchange value (Fig. 3bd and Supplementary Figs. 3l 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 from 25 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 housekeeping gene lmn-1 as a reference. calcium imaging analysis. In vivo calcium imaging was performed according to previously reported methods9,28, but with some modifications. To monitor the temperatureevoked 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 Peltierbased 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 DualView optics system (Molecular Devices). Cyan fluorescent protein (CFP; F480) and yellow fluorescent protein (YFP; F535) images were simultaneously captured by an electronmultiplying chargecoupled device camera, C910013 ImagEM (Hamamatsu Photonics). For calcium imaging during transient upanddown

warming and stepwise warming, images were taken with a 500ms exposure and 1 1 binning. For Figure 8e, to protect the probe from photobleaching during measurement, each image was taken with a 100ms excitation pulse at a 1Hz frame rate using a pulsegeneratorequipped imaging apparatus (SG4115, Iwatsu). The temperature on the agar pad was monitored by a thermometer sys tem, DCM20 (Tokai Hit and Hamamatsu Photonics). Fluorescence intensities of F535 and F480 were measured using the MetaMorph imaging analysis system (Molecular Devices). Statistical analysis. The statistical analysis for Figures 4ei and 6e was performed using twoway analysis of variance followed by post hoc analysis using the Fishers protected least significant difference test, whereas that for Figures 2ce, 3be, 5d, 7ad and 8ad and Supplementary Figure 3lo was conducted using ttests. Differences were considered to be significant when P < 0.05.
37. Mello, C.C., Kramer, J.M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 39593970 (1991). 38. Von Stetina, S.E. et al. Cell-specific microarray profiling experiments reveal a comprehensive picture of gene expression in the C. elegans nervous system. Genome Biol. 8, R135 (2007). 39. Watson, J.D. et al. Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system. BMC Genomics 9, 8497 (2008). 40. Irizarry, R.A. et al. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 31, e15 (2003). 41. Murphy, C.T. et al. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424, 277283 (2003).

2011 Nature America, Inc. All rights reserved.

nature neurOSCIenCe

doi:10.1038/nn.2854