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Introduction

Organisms on Earth have evolved an internal timekeeping system, or circadian clock (circa = about, diem = day), that allows them to both respond to and predict changes in the 24-h environmental day. Much has been learned about the genes involved in this precise, 24-h molecular timekeeping mechanism in the fruit fly Drosophila melanogaster (for a recent review see [1]). The fly clock is composed of intracellular feedback loops: The proteins Clock (CLK) and Cycle (CYC) activate transcription of period (per), timeless (tim), vrille (vri), and PAR-domain protein 1 (Pdp1). Subsequently, proteins encoded by the latter four genes either suppress or activate CLK and CYC [2–8]. Feedback in these regulatory loops is thought to oscillate due to timed changes in the stabilities and subcellular localizations of component proteins, especially Period (PER) and Timeless (TIM) [9,10].

The fly molecular clock is aligned to the environment through Zeitgebers (“time givers”), the most notable being the daily light/dark cycle. This is mediated by the light-dependent degradation of the TIM protein [11,12]. Cryptochrome (CRY), a blue light photoreceptor in the family of flavoproteins, has been shown to associate with TIM during the light phase of the circadian day, resulting in ubiquitination and degradation of TIM by the proteasome and ultimately relieving inhibition of CLK-mediated transcription [13–15]. In addition, a second pathway of light entrainment in the pacemaker neurons is defined by signals from visual organs that may impact TIM in a CRY-independent manner [13,16].

Light is the best understood Zeitgeber, but other factors, such as daily changes in temperature [17–20] and social behavior [21], can act as inputs to the fly circadian clock. Although the fly clock is temperature-compensated over a wide range of constant physiological temperatures, it has been known for several decades that eclosion in Drosophila pseudoobscura can entrain cycling temperature changes [20]. Further, it was shown in this species that temperature step-ups, step-downs, and pulses result in accompanying phase shifts in behavior [22]. Locomotor activity behavior in D. melanogaster can be entrained to temperature cycles of as little as 3 °C [18]. The locomotor activity rhythms of arrhythmic clock mutants (per⁰, tim⁰¹, Clk^Jrk, cyc⁰) can be driven by temperature [19]. However, these “clock-less” mutants do not truly entrain as there is no anticipation of the temperature transitions, and rhythmicity does not persist when they are released into constant conditions. Interestingly, the locomotor activity of wild-type flies can be entrained to temperature cycles during constant light, a condition that would normally result in arrhythmicity [17,19,23]. There is anticipation of temperature transitions, but as with the arrhythmic clock mutants, locomotor activity behavior becomes arrhythmic when the temperature cycle is removed.

Molecularly it has been shown that short, high temperature heat pulses result in rapid downregulation of both PER and TIM proteins [24]. This results in a phase delay if the heat pulse is administered in the early night. However, a heat pulse given in the late night does not result in a phase advance, as is the case with a light pulse given at this time. This is thought to be due to a rapid increase in PER and TIM production after the initial downregulation, ultimately resulting in a constant period [24]. It is not clear if the molecular responses triggered by abrupt heat pulses also play an important role in the entrainment of the molecular clock circuits to environmental temperature cycles.

PER and TIM proteins oscillate during temperature entrainment in constant darkness (dark/dark or DD), and these oscillations are maintained during constant conditions following entrainment [13]. It thus follows that temperature acts on at least some of the same molecular components of the circadian clock as light. Temperature cycles can also drive PER and TIM oscillations during constant light, a condition that, as mentioned earlier, normally results in behavioral and molecular arrhythmicity [17,23].

In this report, we examine temperature as a Zeitgeber for the circadian clock and ask whether information from temperature is relayed through the same molecular circuits as light. In nature, the maxima and minima of solar irradiation and environmental temperature are offset (Figure 1) [25]. Sunrise generally coincides with the coolest part of the day and maximum solar irradiance at noon precedes the temperature maximum in the late afternoon. The divergent phases and waveforms of the environmental light and temperature profiles could in principle be represented by separate Zeitgeber-specific oscillators, or they could be integrated by a single oscillator capable of synchronizing to both photocycles and thermocycles.

Figure 1. Relationship between Daily Light and Temperature Cycles

Under natural environmental conditions, air temperature (purple line and blue = cold/red = warm scale) shows a more gradually changing profile than solar irradiance (gray line and black = dark/white = light scale). Incoming solar energy affects air temperature indirectly via the Earth's surface and this causes a lag between the profiles for sunlight and air temperature, with the temperature maximum and minimum occurring in the late afternoon and just before sunrise, respectively. These profiles are representative of calm, clear days; the lag in the environmental profile can be shortened or lengthened depending on factors such as cloud cover and wind (adapted from [25]).

doi:10.1371/journal.pgen.0030054.g001

By generating genome-wide transcriptional profiles during temperature cycles and subsequent constant conditions, we show that there are two distinct responses to temperature: a clock-independent, temperature-driven response and a clock-dependent, circadian response. Temperature-entrained circadian transcript profiles show a much higher degree of overlap with light-entrained circadian transcript profiles than do temperature-driven responses. Further, the mutual phase relationships among transcripts oscillating in response to both photo- and thermocycles are maintained in both conditions. Thus, many features of the circadian expression program emerge independently from the precise nature of the environmental Zeitgeber. Moreover, the molecular phases associated with separate photocycle and thermocycle entrainment suggest synergistic synchronization by the environmental light and temperature profiles found under most natural conditions.

Results

Genome-Wide Expression Profiles Indicate Two Distinct Responses to Temperature Entrainment

Genome-wide transcript profiles for the heads of temperature-entrained flies were determined in four 12-point time course experiments conducted in constant darkness; three time courses (two for wild-type and one for arrhythmic mutant tim⁰¹ flies) consisting of a day-long 12-h 18 °C/12-h 25 °C (cold/ambient or CA) thermocycle plus a subsequent day of constant 25 °C (ambient/ambient or AA) and one time course (for wild-type flies) spanning the first two days of constant conditions (see Materials and Methods). In order to identify high-confidence, 24-h periodic gene expression, we compared the distribution of oscillatory statistics obtained from Affymetrix high-density oligonucleotide arrays to a permutation null model (see Materials and Methods; [26,27]). For each probe set on the arrays, we determined the 24-h spectral power and the probability of observing an equivalent or higher score from a genome-wide set of randomly permuted profiles. Analysis data are made available at http://biorhythm.rockefeller.edu. We then determined the number of selected 24-h periodic genes as a function of the threshold p-value or the associated false discovery rate (FDR) as illustrated by the graphs in Figure 2. These analyses were performed for various combinations of the new datasets representing temperature entrainment and of previously described datasets [27–29] representing light entrainment. Our analysis method emphasizes coherence of phase and period length but does not penalize inter-experimental variation in amplitude [26]. We have used this method to demonstrate that analysis of combinations of independently obtained time course microarray datasets representing the same or a similar environmental protocol allows for the detection of periodic expression programs with improved resolution [26,27]. The quantitative differences between the observed 24-h periodic expression programs are best visualized in Figure 2A, where an arithmetic scale is used.

Figure 2. Comparison of Temperature-Driven, Light-Driven, Light-Entrained Circadian and Temperature-Entrained Circadian Daily Expression Programs

The number of selected rhythmic transcripts for the indicated time course microarray datasets was determined as a function of the estimated FDR (A) or as a function of the threshold applied for the probability associated with 24-h spectral power (B–E). See Materials and Methods for a detailed description of the statistical procedures. Temperature-entrained and light-entrained circadian regulation are represented by 4-d wild-type (4x wt) datasets collected under constant conditions (25 °C in the dark) following temperature entrainment (AA) or light entrainment (DD) in (A) and (D), whereas temperature-driven and light-driven regulation are represented in (A) and (C) by datasets combining 2 d of wild-type plus 1 d of arrhythmic mutant data (2x wt + 1x tim⁰¹) collected in the presence of an 18 °C/25 °C thermocycle in the dark (CA) or in the presence of a 12-h light/12-h dark cycle at 25 °C (LD). In addition, datasets are included in (A) and (B) that consist of 2 d of wild-type CA or LD data representing a combination of temperature-driven and temperature-entrained circadian regulation or light-driven and light-entrained circadian regulation, respectively. Finally, 6-d wild-type datasets are considered in (A) and (E) that combine 2x CA and 4x AA or 2x LD and 4x DD data and represent temperature-entrained circadian regulation or light-entrained circadian regulation with some influence from temperature-driven or light-driven responses as well. Note that the y-axis scale in (A) is arithmetic but in (B–E) is geometric. The bracketed numbers in (A) indicate the number of selected transcripts for each of the analyses at FDR ~0.2. Comparisons of the 24-h periodicity for these various datasets indicate that the circadian expression programs found in response to temperature or light entrainment have very similar properties, but that there is a large clock-independent temperature-driven expression program that clearly has a more global effect than circadian or light-driven regulation.

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It is clear that a 2-d wild-type dataset representing a CA environmental temperature cycle shows a much broader impact on global 24-h periodicity than an equivalent dataset representing a 12-h light/12-h dark (LD) cycle (e.g., 326 versus 42 periodic transcript profiles at FDR 0.2; see Figure 2A and 2B). This result could be explained by either a temperature-driven, clock-independent effect or by a thermocycle-specific, clock-dependent effect. Additional comparative analyses help to distinguish between these two possibilities. First, consider expanded 3-d versions of the CA and LD datasets that each also include a 1-d time course in the same format obtained from arrhythmic tim⁰¹ flies. Given that both behavioral and molecular circadian rhythms appear to be abrogated by the tim⁰¹ mutation [27,30], the additional time course data may represent temperature- or light-driven, but not clock-dependent, expression profiles. The enhanced difference in 24-h periodicity between thermocycle and photocycle conditions that results from inclusion of the tim⁰¹ data (e.g., 939 versus 72 periodic transcript profiles at FDR 0.2; Figure 2A and 2C) indicates that most of the thermocycle-associated transcript rhythms are simply temperature-driven independently from the clock. Second, compare the properties of 24-h periodic transcript profiles of 4-d datasets representing the same constant conditions (25 °C and constant darkness) after either temperature entrainment (AA) or light entrainment (DD). The circadian programs detected after entrainment to temperature and light have very similar properties (Figure 2A and 2D; see also the section Defining a Set of Clock-Dependent Transcripts, below), supporting the hypothesis that the increased 24-h periodicity found in the context of an environmental temperature cycle is due to clock-independent temperature-driven regulation rather than to circadian rhythms that specifically require temperature entrainment.

The broad temperature-driven response observed in the presence of a temperature cycle could in principle interfere with the circadian expression program. This issue is addressed by analysis of a 6-d dataset combining 2 d of temperature entrainment and 4 d of subsequent constant conditions (CA/AA) and an equivalent 6-d dataset representing light entrainment and subsequent constant conditions (LD/DD). Both of these programs indicate more high-quality daily transcript oscillations than are found separately for the 2-d (CA or LD) or 4-d (AA or DD) subsets (Figure 2A and 2C–2E). Even the sum total of high-quality daily transcript oscillations from the 2-d and 4-d subsets is considerably less than the number observed for the integrated 6-d sets (e.g., 33 for 2x CA plus 27 for 4x AA versus 212 for 2x CA/4x AA and 13 for 2x LD plus 20 for 4x DD versus 75 for 2x LD/4x DD at FDR 0.05, Figure 2A and 2C–2E). This suggests that, in general, circadian expression profiles are not dramatically altered by temperature-driven (or light-driven) effects (Figure 2A, 2B, 2D, and 2E).

Comparison of the 24-h periodicity found in the 6-d wild-type CA/AA and LD/DD sets versus the 4-d wild-type AA and DD sets and the 3-d wild-type plus tim⁰¹ CA and LD sets further illustrates the magnitude of the temperature-driven response (Figure 2A, 2C, and 2E). There is no reason to assume that purely circadian expression profiles are more prevalent or prominent in CA versus LD conditions, yet inclusion of 2 d of CA data with the AA set has a bigger impact on 24-h periodicity than the inclusion of 2 d of LD data with the DD set, suggesting that temperature-driven regulation may be responsible. In addition, although more extensive 24-h periodic expression is generally detected for larger datasets [26,27], the daily expression program found for the 3-d wild-type plus tim⁰¹ CA dataset is comparable in size to that for the 6-d wild-type CA/AA dataset. In contrast, the 6-d wild-type LD/DD dataset shows much more extensive 24-h periodicity than the 3-d wild-type plus tim⁰¹ LD dataset, indicating that the presence of a daily thermocycle is the primary determinant of the number of observed 24-h transcript oscillations.

Taken together, these analyses suggest that the circadian expression programs entrained by light entrainment and temperature entrainment have similar properties, whereas an environmental thermocycle directly evokes a global expression response that is considerably broader than that found for light-dependent or clock-dependent regulation.

Defining a Set of Temperature-Driven Transcripts

A core set of the most robustly temperature-driven transcripts was identified based on wild-type and tim⁰¹ thermocycle expression profile data. In order to be included, transcripts had to meet several noise filters and show a highly significant 24-h Fourier component as well as a significant 24-h autocorrelation (see Materials and Methods). The phasegram for the resulting set of 164 temperature-driven transcripts in both wild-type and tim⁰¹ flies is shown in Figure 3A. The majority of these transcripts respond with a simple pattern of either activation during the warm phase and repression during the cold phase or vice versa. Further, most of this response is lost in constant conditions following temperature entrainment (Figure 3A) or during and after light entrainment (unpublished data), emphasizing that the response is driven and not circadian.

Figure 3. Phases of the Temperature-Driven Transcripts

(A) Phasegrams for transcripts from wild-type (wt) and tim⁰¹ flies in CA/AA are shown. Columns correspond to time points, and transcript profiles are represented by rows. Rows are ordered according to the estimated peak phase of the transcript profiles in CA conditions. Expression values represented by increasingly bright shades of magenta and cyan indicate, respectively, upregulation and downregulation relative to the experimental average (indicated by light gray).

(B) Histogram showing the estimated peak phases (ZT h) of the temperature-driven transcripts. The red, blue, and violet bars in (A) and (B) indicate the warm, cold, and subjective cold phases, respectively.

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Figure 3B shows the average peak phases of the temperature-driven transcripts across data from wild-type and tim⁰¹ flies during temperature entrainment. Two trends are obvious from this analysis: (1) temperature-driven oscillations tend to peak around the middle of either the cryophase or the thermophase, and (2) more transcripts peak during the cryophase than during the thermophase. If the term CA0 is assigned to the onset of the cold temperature and CA12 to the onset of the ambient temperature, the majority of the transcripts have a phase of either CA5–8 (toward the middle of the cryophase) or CA18–20 (toward the middle of the thermophase). Given our use of sine wave fits to estimate peak phases, the observed phase distribution is consistent with a majority of the expression profiles being directly positively or negatively temperature-driven with relatively little delay. Approximately three-quarters of the temperature-driven profiles peak in the cryophase, but the functional relevance of this preference is not directly obvious.

The temperature-driven transcripts are representative of diverse biological functions (Table S1), including carbohydrate, amino acid, lipid/fatty acid, one-carbon compound, nucleic acid, folate, and steroid metabolism, as well as transport, signal transduction, development, behavior, protein translation, protein modification, protein folding, proteolysis, defense/immune response, muscle contraction, cytoskeleton, and exoskeleton. In comparison with a set of 143 predicted temperature-entrained circadian transcripts that is described in more detail below (see Figure 4 and Materials and Methods), the temperature-driven transcripts show a higher frequency of functions associated with transport, transcription, translation, development, proteolysis, and protein folding, but a lower frequency of functions associated with circadian behavior and carbohydrate metabolism.

Figure 4. Phases of the Clock-Dependent Transcripts

The transcripts from wild-type flies in CA/AA are shown by phase in the same format as Figure 3. Columns correspond to time points, and transcript profiles are represented by rows. Rows are ordered according to the estimated peak phase of the transcript profiles across the CA/AA data. Expression values represented by increasingly bright shades of magenta and cyan indicate, respectively, upregulation and downregulation relative to the experimental average (indicated by light gray). The red, blue, and violet bars above the phasegram indicate the warm, cold, and subjective cold phases, respectively.

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Defining a Set of Clock-Dependent Transcripts

All available microarray time courses from wild-type flies during and after temperature entrainment were used to define a set of temperature-entrained circadian transcripts. In order to be included, transcripts had to meet several noise filters and show a highly significant 24-h Fourier component as well as significant 24-h autocorrelation (see Materials and Methods) across the complete 2x CA/4x AA dataset. In order to avoid transcript oscillations that were merely temperature-driven, it was required that they also show a significant 24-h Fourier component and exceed background noise in an analysis of AA data only (see Materials and Methods). The resulting set of 143 temperature-entrained circadian transcripts is presented in a phasegram (Figure 4), and the functions associated with these transcripts are described in Table S1. As noted above, the set of temperature-entrained circadian transcripts shows some differences in its functional representation relative to the set of temperature-driven transcripts. Perhaps the most remarkable functional enrichment among temperature-entrained circadian transcripts is found for the takeout (to) gene family, which has been proposed to contribute to courtship behavior, starvation response, and olfaction [31,32]. Seven of the 21 members of this gene family show a strong temperature-entrained circadian expression component, whereas a robust temperature-driven response (represented by oscillating expression in a temperature cycle in tim⁰¹ flies) is only found for one gene, which happens to also show circadian regulation (Table S1). We also defined a set of 172 light-entrained circadian transcripts by applying similar selection criteria to the results from previous analyses of all available LD/DD microarray time course data [27] (see Materials and Methods). The overlap between the two datasets (49 transcripts) is highly significant and involves about a third of the transcripts in each set (Figure 5A and Table S1), which is considerably more than, for example, the overlap of either set with the set of 164 temperature-driven transcripts from Figure 4 (overlap of 22 with the temperature-entrained and 13 with the light-entrained circadian transcripts). The fact that two-thirds of the predicted temperature-entrained circadian transcript profiles are not represented in a stringently selected set of light-entrained circadian transcript profiles does not mean that they do not show significant light-entrained circadian oscillations. The degree of overlap between the independent selections is quite sensitive to factors such as choice of selective cut-off criteria and reproducibility of the relative rankings of circadian transcripts. Nevertheless, the fact that the overlap is incomplete could indicate that there are both light- and temperature-specific circadian transcript oscillations. To examine the relative importance of such Zeitgeber-specific entrainment, we tested whether the overlap between two independent 4-d DD datasets was substantially larger than that between each of the two DD sets and our 4-d AA dataset; the results are illustrated in Figure 6. The two independently detected DD circadian expression programs did not obviously share more transcripts with each other than with the AA circadian expression program. There is, therefore, no evidence for widespread temperature-specific or light-specific circadian expression. To further investigate this issue, six potential temperature-specific and three potential light-specific circadian transcript profiles were verified using northern blots. All six of the potential temperature-specific circadian transcripts show circadian oscillations in LD/DD (Figure S1A), whereas none of the three transcripts with a predicted light-dependent circadian response exhibited clock-dependent regulation in response to temperature entrainment (Figure S1B). These results are consistent with the notion that most, but not all, light-entrained circadian transcripts are also entrained by an environmental temperature cycle.

Figure 5. Overlap and Mutual Phase Relationship between Those Transcripts Oscillating in CA/AA and LD/DD

(A) Wild-type transcripts oscillating in response to both photo- and thermocycles are shown by phase in the same format as Figure 3. Columns correspond to time points, and transcript profiles (with gene names listed to the right) are represented by rows. Rows are ordered according to the estimated peak phase of the transcript profiles across the LD/DD data. Expression values represented by increasingly bright shades of magenta and cyan indicate, respectively, upregulation and downregulation relative to the experimental average (indicated by light gray). The red, blue, violet, white, black, and gray bars above the phasegram indicate the warm, cold, subjective cold, light, dark, and subjective light phases, respectively.

(B) The phases of transcripts oscillating in CA/AA are “advanced” (relative to the onset of the respective Zeitgeber) by about 6 h as compared to LD/DD. The bars above and to the right of the plot denote the entrainment scheme. Each red square on the plot corresponds to a transcript, with its LD/DD phase indicated on the x-axis and its CA/AA phase indicated on the y-axis. The data were fit to a regression line with slope 1 as indicated.

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Figure 6. Overlap Analysis of Independently Determined Circadian Expression Programs after Light or Temperature Entrainment

Three independent 4-d wild-type (4x wt) datasets collected under constant conditions (25 °C in the dark) following temperature entrainment (AA) or light entrainment (DD₁ and DD₂) are indicated. See Materials and Methods for details. The number of selected rhythmic transcripts for each of these datasets is graphed as a function of the threshold applied for the probability associated with 24-h spectral power (upper three lines). The size of the pairwise overlap between the circadian transcript selections as a function of the selective p-value is also indicated (lower three lines).

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Environmental Photocycles and Thermocycles Entrain the Circadian Clock to Similar Molecular and Behavioral Phases

Identification of a set of transcripts that show especially robust circadian regulation in response to both entrainment by light and entrainment by temperature allowed us to directly compare the circadian phases dictated by these two Zeitgebers. When all available LD/DD and CA/AA time course microarray data for this set is ordered in a phasegram according to the observed light-entrained phase, it becomes apparent that temperature-entrained phases are directly correlated with independently determined light-entrained phases, suggesting that mutual phase relationships among circadian transcripts may be preserved under entrainment of either light or temperature (Figure 5A). When estimated light-entrained and temperature-entrained peak phases are directly plotted against each other, it becomes apparent that their relationship can be described by a linear function that represents a fixed phase offset (Figure 5B). When the light-entrained phase is determined relative to lights-on and the temperature-entrained phase is determined relative to the onset of the thermophase, the value of the offset is ~6 h. In the context of natural environmental conditions (Figure 1), however, the light/dark and temperature cycles are aligned differently, with the temperature minimum occurring just before dawn and the temperature maximum delayed relative to the time of maximum solar irradiance. It would be, therefore, more appropriate to assign temperature-entrained phases relative to the time of subjective dawn (the middle of the cryophase). In this context, the observed temperature-entrained and light-entrained molecular phases would, in fact, roughly coincide, indicating cooperative entrainment by environmental cycles in light and temperature under most natural conditions. Convergence of photic and thermal entrainment is also observed at the behavioral level (Figure S2). During LD cycles, flies are preferentially active around the dark-to-light and light-to-dark transitions, with a siesta in the middle of the day. Although the sudden changes in environmental light at the transitions directly elicit behavioral startle responses in a clock-independent manner, a functional clock is required for control of a major circadian activity component at dusk and a minor circadian activity component at dawn (e.g., [18]). During temperature entrainment, the outlines of a similar pattern of clock-dependent activity can be recognized with onset of dawn-associated activity occurring during the last half of the cryophase and dusk-associated activity coinciding with the middle of the thermophase (Figure S2).

We further examined the cooperative effects of light and temperature on entrainment of locomotor activity behavior. We entrained flies to an LD cycle and then released them into constant darkness in which a temperature cycle was given either “in phase” (i.e., light onset precedes onset of the thermophase by 6 h) or “out of phase” (i.e., thermophase onset precedes light onset by 6 h). When light and temperature are given in phase, the average time of activity offset after 5 d of temperature is the same as the previous light phase (Zeitgeiber time [ZT] 13.23 ± 1.32 versus ZT 13.98 ± 0.95; Figure 7). However, when light and temperature are given out of phase, the average time of activity offset gradually advances over 5 d of temperature to approximately 10 h earlier than that measured in LD (ZT 3.70 ± 2.90 versus ZT 13.46 ± 0.56; Figure 7B and 7C). Thus, the temperature- and light-entrained circadian behavioral phases show essentially the same relationship as the molecular phases that we discussed above. Under “natural” environmental conditions (i.e., when light onset and offset coincide with the middle of, respectively, the cryophase and thermophase) both molecular and behavioral rhythms are entrained cooperatively by temperature and light.

Figure 7. Cooperative and Antagonistic Effects of Light and Temperature on Locomotor Activity Behavior

(A–B) The average locomotor activity of a number of flies (A, n = 23; B, n = 18) is represented in each panel. The data are double plotted for visual continuity. Flies were recorded for 4 d in an LD cycle, as indicated by the open and closed bars above the panels, respectively. The flies were then released into DD in which a 25 °C/18 °C temperature cycle was given “in” phase (A, onset of light precedes the onset of warm temperature by 6 h) or “out” of phase (B, onset of warm temperature precedes the onset of light by 6 h).

(C) The average times of activity offset relative to the initial LD cycle and the associated standard deviation are indicated on each day for flies with in-phase or out-of-phase thermocycles.

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Organization of the Core Clock Components in Temperature Entrainment

The well-characterized core clock transcripts that are known to oscillate under light entrainment (per, tim, Clk, cry, vri, Pdp1ɛ) continue to cycle under temperature entrainment in wild-type flies, while the noncycling core clock transcripts (cyc, dbt) remain constant (Figure 8). In LD cycles, the levels of the core clock transcripts remain at constitutive high or low levels in arrhythmic tim⁰¹ flies [4,30,33–35]. Analysis of the tim, per, Clk, and cry transcripts in arrhythmic tim⁰¹ flies exposed to a temperature cycle, however, reveals underlying temperature-driven responses that do not persist under constant conditions (Figure S3). These temperature-driven oscillations are mostly out of phase with the circadian transcript rhythms observed in wild-type flies (Figure S3). In order to maintain essentially the same circadian expression program for its core components in the presence or absence of an environmental temperature cycle, therefore, the clock has to largely neutralize the direct effects of temperature on their expression.

Figure 8. Expression of the Core Clock Genes in Temperature Entrainment

(A) Northern blots showing expression of the core clock transcripts in wild-type flies in CA and AA. An rp49-specific probe was used as a loading control.

(B) Quantitations from (A). The bars below the northern images and plots denote the entrainment scheme, with red bars indicating 25 °C time points, blue bars indicating 18 °C time points, and violet bars indicating free-run time points taken at 25 °C. The different colored lines in the vri, tim, and Pdp1 plots represent the different transcripts. At least two independent profiles were obtained for each transcript. Peak to trough ratios (P/T) across the entire experiment, probability of circadian rhythmicity (pF24), and predicted circadian phase relative to the onset of the cryophase (phF24) are as follows. per (P/T = 4.8; pF24 = 0.02; phF24 = CA16), tim (smaller transcript P/T = 5.8; pF24 = 0.0001; phF24 = CA18; larger transcript [tim^cold] (P/T = 2.7; pF24 = 0.04; phF24 = CA15), Clk (P/T = 2.7; pF24 = 0.0001; phF24 = CA3), cry (P/T = 4.3; pF24 = 0.04; phF24 = CA8), vri (larger transcript P/T = 2.7; pF24 = 0.15; phF24 = CA13; smaller transcript P/T = 2.2; pF24 = 0.12; phF24 = CA19), Pdp1 (transcripts numbered according to increasing size, Pdp1-1 [not visible on blot] P/T = 3.3; pF24 = 0.18; phF24 = CA21, Pdp1-2 P/T = 4.6; pF24 = 0.02; phF24 = CA20, Pdp1-3 P/T = 3.3; pF24 = 0.003; phF24 = CA17, Pdp1-4 P/T = 3.2; pF24 = 0.0003; phF24 = CA15, Pdp1-5 P/T = 3.4; pF24 = 0.003; phF24 = CA15).

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The phase relationships between core clock transcripts (i.e., per, tim, vri, and Pdp1ɛ oscillate antiphase to Clk and cry) are largely maintained in wild-type, temperature-entrained flies. Entrainment to temperature cycles, therefore, appears to promote the same overall clock organization and function as light entrainment, although with an important distinction that involves per and tim RNA expression. In light entrainment and subsequent free-run, per and tim transcription is tightly coupled at all times (Figure S4). In temperature entrainment, however, per and tim are uncoupled. This is due to both an advance in per expression and a delay in tim expression (compared to free-run; Figure S4). This divergence is absent in constant conditions following temperature entrainment. It is also absent at the protein level (Figure S5). One possible explanation for this discrepancy is that in temperature cycles a shift in the timing of per and tim RNA accumulation may be required to maintain coordinately phased accrual of the PER and TIM proteins.

The advance in per expression could in theory be explained by the effects of a thermosensitive splicing event in the 3′ UTR of per, which is thought to enable flies to seasonally adapt to cold, short days [36–38]. It is important, however, to address this hypothesis experimentally, as alternative splicing of per has not been directly examined in the context of a thermocycle. The delay in tim expression is associated with thermosensitive splicing. A second tim transcript (referred to as tim^cold) is observed during temperature entrainment, especially during the cold phase (Figure 9A). tim^cold is expressed at low levels in light entrainment at 25 °C (Figure 9B), but in light entrainment performed at 18 °C it is the dominant isoform (Figure 9C). Overall tim transcript levels appear to be increased at 18 °C relative to 25 °C by a factor of 1.5–2 (unpublished data).While the canonical shorter transcript of tim oscillates with a phase that differs from that of per during temperature entrainment, tim^cold cycles in phase with per (Figure S4). Total tim transcript levels in the presence of the 18 °C /25 °C thermocycle follow the same pattern as the canonical transcript, albeit at a somewhat lower peak/trough ratio (~3-fold versus ~5-fold). Reverse transcriptase-PCR and northern blot analyses reveal that, in tim^cold, the last tim intron (~850 bp) is retained (unpublished data). This unspliced form of tim has a premature stop codon that would putatively result in a protein about 3.5 kDa smaller than the full-length TIM protein. The missing fragment corresponds to a small piece of the cytoplasmic localization domain [39]. Western blots reveal the presence of two TIM isoforms at 18 °C in light entrainment, the lower of which is downregulated at 25 °C (Figure 9D). Further work will be needed to understand the role of tim alternative splicing.

Figure 9. tim Is Alternatively Spliced at Cold Temperatures

(A–C) An alternatively spliced form of tim RNA (arrows in A–C) is present in wild-type flies in CA/AA (A), especially during the cold phase, and in LD/DD at 18 °C (C). This splice form is less abundant and distinct in flies entrained to LD/DD at 25 °C (B). An rp49-specific probe was used as a loading control for each blot (lower panels).

(D) The alternative transcript contains a predicted premature stop codon and results in a shorter TIM protein isoform (arrow), which can be readily detected in samples collected in LD at 18 °C but is not obvious at 25°C. The horizontal color-coded bars in panels in (A–D) denote the entrainment scheme, with white bars indicating light time points, black bars indicating dark time points, gray bars indicating free-run time points taken during subjective light, red bars indicating 25 °C time points, the blue bar indicating 18 °C time points, and the violet bar indicating free-run time points taken at 25 °C.

doi:10.1371/journal.pgen.0030054.g009

Discussion

Light is the best understood Zeitgeber for the circadian clock, but many organisms are also exposed to daily changes in temperature in their natural environments that may influence their clocks. In this study, we describe entrainment of molecular and behavioral circadian rhythms by environmental thermocycles. Genome-wide expression profiles of transcripts during temperature entrainment and subsequent constant conditions in both wild-type and arrhythmic tim⁰¹ backgrounds were generated in order to analyze the role of temperature cycles on gene expression in the fly. Unlike in light entrainment, where the magnitude of the overall transcriptional response in the presence or absence of a photocycle is largely maintained, there is a dramatic difference in transcriptional responses in the presence or absence of a thermocycle. Our results indicate, therefore, that temperature-driven responses may be a major determinant of daily fluctuations in gene expression. Because the day-to-day variability in temperature profiles found in most natural climates would negatively impact the stability of temperature-driven daily expression rhythms, the observed temperature-driven regulation of gene expression may primarily serve to mediate short-term responses to changes in temperature. Whereas transcription appears to be modified globally by temperature cycles, there are a limited number of transcripts that continue to oscillate in constant conditions following temperature entrainment. Thus, temperature cycles elicit both a clock-independent, temperature-driven response and a clock-dependent, circadian response (Figure 10).

Figure 10. Model of How Information from Light and Temperature Are Processed by the Fly Circadian Clock

Information from light and temperature, which is naturally out of phase, is relayed through the appropriate sensors to the clock. In the absence of photic or thermal input, the clock can predict when the fly would have seen light and dark or warm and cold, respectively. When both Zeitgebers are present, they are integrated by the clock to generate meaningful phases of transcription (green). Independently of the clock, light can directly affect (through light sensors) the transcription of a small number of genes (yellow), whereas temperature can drive the expression of a larger number of transcripts (blue). Although some potentially relevant thermal sensors have been reported based on genetic evidence [17], it is unclear to what extent they are involved in determining temperature-entrained and temperature-driven transcript rhythms at a genome-wide level. There are also transcripts that are dually regulated by the clock and light (orange) or temperature (purple), which may be important for processes such as seasonal adaptation.

doi:10.1371/journal.pgen.0030054.g010

A set of clock-independent, temperature-driven transcript profiles was defined by analyzing both the wild-type and tim⁰¹ data in the presence of a thermocycle. Since these transcripts are directly driven by a thermocycle, their behavior should be the same in the presence or absence of a functional clock. However, due to the inclusion of wild-type data, this set could, in theory, include transcripts that are both temperature-driven as well as clock-dependent. Dual regulation by both the circadian clock and temperature or light may allow for seasonally modulated daily transcript rhythms. A set of dually light- and clock-regulated transcripts was recently identified in the context of an LD cycle [27] (Figure 10). In an arrhythmic clock mutant, these transcripts are simply induced or repressed in response to light. In wild-type flies, however, input from both light and the clock results in transcript profiles combining light-driven and circadian expression components. In the present study, we found significant overlap between the set of transcripts that show temperature-driven regulation and the set of either temperature-entrained or light-entrained circadian transcripts. For example, 22 transcripts are part of both the temperature-driven set described in Figure 3 and the temperature-entrained circadian set described in Figure 4, indicating that there is, indeed, a class of transcripts with both circadian- and temperature-driven regulation, whose circadian- and temperature-driven expression components are well aligned. Moreover, our in-depth analysis of core clock transcript profiles has uncovered a second class of dually temperature- and clock-regulated transcripts, whose circadian- and temperature-driven expression components favor opposite peak expression phases. Comprehensive identification of transcripts belonging to this second class would require collection of additional time course microarray data, so that thermocycle-associated periodicity and phase can be determined separately for arrhythmic mutant flies.

We also defined a set of clock-dependent, circadian transcripts in this study. This set of temperature-entrained transcripts shows a highly significant overlap with those transcripts that oscillate in response to a photocycle. Further, the light- and temperature-entrained phases of these transcripts roughly coincide in the context of natural environmental conditions. These observations indicate that in the fruit fly a single TIM-dependent transcriptional clock mechanism produces a core circadian expression program that can be synchronized to different environmental Zeitgebers. Although the global properties of the light-entrained and temperature-entrained circadian expression programs are very similar, we have identified three examples of genes that only exhibited circadian regulation in response to an environmental light/dark cycle. This novel type of regulation may represent a previously unrecognized functional interaction between light-sensing pathways and the circadian clock. It is also possible, however, that the apparently light-specific circadian genes are not completely insensitive to temperature entrainment, but merely require higher thermocycle amplitude or a prolonged period of entrainment.

Differential Regulation of per and tim

Transcriptional regulation of per and tim appears to be different in light and temperature entrainment. Whereas in light entrainment per and tim RNA expression is tightly coupled at all times, in 18 °C/25 °C temperature entrainment per RNA levels peak before tim RNA levels. This is a result of a temperature-induced advance in per expression and delay in the expression of the predominant tim transcript. Differences in per and tim regulation have been suggested based on the observation that these transcripts show different rates of degradation in response to a light pulse in the context of the long period mutant tim^ul [40]. In addition, while at lower temperatures per expression is upregulated in LD and DD, tim has been reported to be downregulated in LD and barely oscillatory in DD [38]. Further, while the phases of both per and tim appeared advanced at lower temperatures, the advance in per was interpreted as a result of faster accumulation, while the advance in tim was thought to represent more rapid degradation [38]. It has also very recently been reported that tim, but not per, transcript levels are upregulated in response to light pulses at cold temperatures [41]. It is noteworthy, however, that the probe used in several previous studies [24,38,41] to evaluate tim transcript expression with RNase protection assays may not have efficiently detected the tim^cold isoform since it spans the exons flanking the intron maintained in tim^cold. This issue is illustrated by quantitation of the data in Figure 9C, which confirms the predicted decrease of tim transcript in the first day of DD at 18 °C [38] for the predominant isoform, but not for tim^cold, which shows a more prominent peak in expression (unpublished data). Additional analyses that take into account the contribution of the tim^cold isoform will, therefore, be needed to complement previous studies in order to more fully explore tim transcript responses.

One of the factors involved in the reported differential expression of per and tim may be the alternative splicing of both transcripts. Much of the recent molecular work on temperature and the circadian clock has focused on the alternative splicing of an 89-bp intron in the 3′ UTR of per, an event thought to be important in seasonal adaptation [36–38]. Short, cold days lead to increased amounts of the spliced per variant, resulting in an earlier increase in PER protein abundance and an advanced phase of locomotor activity. Warmer temperatures result in less of the spliced variant, especially during the day. This appears to be a clock-dependent effect that results in the fly moving its behavior to the later (cooler) part of the day. Thus, per splicing allows the fly to adapt to changes in both temperature and photoperiod by regulating the amount of available PER protein. per alternati