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.

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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.

https://doi.org/10.1371/journal.pgen.0030054.g002

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.

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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.

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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.

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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.

https://doi.org/10.1371/journal.pgen.0030054.g005

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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.

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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.

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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.

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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.

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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 alternative splicing is thought to be important in seasonal adaptation, as long photoperiods counteract the cold-induced behavioral advances by delaying the accumulation of TIM, in turn rendering prematurely produced PER unstable. Thus, the fly is able to integrate information from both light and temperature to generate behavior that is aligned to the environmental day. Regulation of per splicing in the presence of an environmental temperature cycle as compared to constant temperature needs to be investigated.

Temperature-dependent alternative splicing of tim is described here. At 18 °C, the last intron of tim is preferentially retained, resulting in a premature stop codon and a truncated protein. Although the expression of the predominant tim transcript is delayed relative to per, tim^cold cycles in phase with per. The differential expression of the two tim transcripts could reflect temperature-dependent control of splicing or of the stability of one of the splice forms. We are still in the process of ascertaining the functional significance of the production of tim^cold transcript. It does, however, appear that the alternative splicing and differential regulation of per and tim are responsible for finely tuning the clock in response to changing environmental conditions, thus adding an additional level of complexity to the clock.

Two Zeitgebers, One Clock

Different groups of clock-bearing cells in the fly have been shown to regulate different rhythmic processes. For example, locomotor activity and eclosion rhythms, arguably the best-characterized rhythmic behaviors in Drosophila, require the ventral lateral neurons (LN_vs) and the neuropeptide, Pigment Dispersing Factor [42–44]. Cyclic olfactory responses do not depend on the LNs or Pigment Dispersing Factor, but instead depend on the antennal neurons [45,46]. Egg-laying rhythms also appear to be regulated independently of the LN_vs and Pigment Dispersing Factor [47]. Thus, the image of the circadian clock as a single entity is transforming into a more complex model.

A system of two coupled oscillators was proposed for the Drosophila clock almost 50 y ago [48]. In this model, the master or A oscillator is autonomous, light-sensitive, and temperature-compensated. The slave or B oscillator, which is coupled to and driven by A, is responsive to temperature but not light. The evidence for this two-oscillator model came from the different responses in eclosion rhythms to light and temperature. Whereas light pulses administered at different times of day resulted in steady-state phase advances or delays, the phase changes resulting from temperature pulses were transient. The researchers concluded that the steady-state phase changes in response to light were a result of the eventual realignment of the A oscillator to the light signal. The transient responses to temperature pulses were proposed to be a result of temporary temperature-induced disturbances in B, with the return to the previous phase reflecting the A oscillator's resumption of control over B.

Coupled oscillators have been proposed in a variety of organisms [49]. For example, different genes are expressed with different period lengths in some Synechoccus mutant backgrounds [50], and bioluminescence rhythms in Gonyaulax have been shown to be regulated by two oscillators that respond to different wavelengths of light [51]. In Neurospora, strains carrying null alleles of frequency (frq), white collar-1 (wc-1), or white collar-2 (wc-2) still show a conidial banding rhythm. Although the “FRQ-less oscillators” [52] responsible for these rhythms have lost most characteristics of a circadian clock, they can be entrained by Zeitgebers such as temperature cycles [53,54], rhythms in nitrate reductase activity [55], and transfer from light to dark [56]. This suggests that FRQ-less oscillators are slave oscillators, requiring the master FRQ-dependent clock to produce stable circadian rhythms, yet with the ability to function independently of the master clock in a non-circadian manner [57].

A system of coupled oscillators has recently been demonstrated in the regulation of the morning and evening peaks of locomotor activity in the fly [58–60]. The morning oscillator requires the presence of the LN_vs, while the evening oscillator requires the dorsal lateral neurons. It was further shown that the evening oscillator is set by the morning oscillator by generating flies in which the morning and evening oscillators have different free-running periods [60]. However, despite the parallels to Pittendrigh's original model, there is no published evidence that these or other oscillators would differentially respond to temperature, as opposed to light, as a Zeitgeber. So while it appears there is a multicellular clock network in Drosophila that is reflected by coordinate yet independently regulated outputs, the data presented here suggest that the response to multiple inputs, such as light and temperature, would still be integrated by a single autonomous clock mechanism (Figure 10). In today's jargon we would describe Pittendrigh's B oscillator as a circadian output pathway that can show direct clock-independent responses to temperature.

The following observations support the hypothesis of a single, integrative transcriptional oscillator. First, the same set of core clock components (including PER, TIM, CLK, and CYC) appears to be required for producing both light-entrained and temperature-entrained oscillations [18,19]. The global transcriptional signatures of arrhythmic tim⁰¹ flies that we found after thermocycle treatment resemble those found after photocycle treatment [27] and do not exhibit obvious circadian rhythms. In addition, our results confirm the absence of circadian oscillations for core clock gene transcripts in the tim⁰¹ fly heads. Second, it is likely that the set of transcripts entrainable by thermocycles is closely related to the set of transcripts entrainable by light. Although we cannot formally exclude the existence of circadian rhythms that specifically require temperature entrainment, we have found none so far. Third, the phases of the transcripts that oscillate in response to both photo- and thermocycles maintain the same mutual phase relationships after entrainment by light or temperature. The phase observed at the onset of the thermophase is systematically advanced by about 6 h relative to the phase at the onset of light. Given the size of the delay that is commonly found between the environmental profiles for temperature relative to that of daylight, these results indicate cooperative entrainment by light and temperature under common natural circumstances. A response to temperature would be well integrated with the expected light cycle were it also supplied, and vice versa. Fourth, the temperature- and light-entrained phases of PER and TIM protein expression [13] (see also Figure S5) reflect the same relationship that we observed for the genome-wide circadian transcript signatures. This observation is consistent with the hypothesis that both light and temperature act via the same PER/TIM-dependent oscillator to generate circadian transcript profiles. Fifth, the entrained phase of locomotor activity behavior appears to follow the molecular circadian phase observed in temperature or light entrainment. Our ability to accurately predict the phase of clock neuron–controlled circadian locomotor behavior based on our analysis of circadian transcript rhythms in a preparation of whole heads, which mostly represents peripheral clock cells, suggests that temperature entrainment just as light entrainment produces similar phases in peripheral clock cells and clock neurons. This result can be verified and extended in a future study by direct examination of the temperature-entrained molecular phase in the various subsets of clock neurons.

In summary, our analyses revealed that thermocycle entrainment and photocycle entrainment produce very similar circadian expression profiles in fly heads, and that under common natural conditions light and temperature are expected to entrain both molecular and behavioral circadian rhythms cooperatively. As pointed out above, our results are in agreement with the notion that a single transcriptional clock is responsible for producing all light-entrained and temperature-entrained circadian rhythms. Nevertheless, we cannot formally exclude the existence of a specialized temperature-entrained oscillator that is coupled to the general transcriptional clock circuits. Such a theoretical temperature-entrained oscillator could have eluded detection in our analyses if it was located outside the head or in a small subset of the cells in the head or if it produced non-transcriptional circadian signals. Elucidation of the mechanisms of thermocycle entrainment will constitute an important next step in defining the temperature-entrained circadian oscillator(s).

Fly strains.

The wild-type strains used were yellow white (y w), Canton S, and cinnabar brown (cn bw); in addition, comparative analyses were performed for y w; tim⁰¹ arrhythmic mutant flies. The flies were raised on standard yeast cornmeal medium.

Time course collection.

For light experiments (described previously; see [27,28]), adult flies were entrained to 12 h of light followed by 12 h of darkness (LD, where ZT0 is lights-on and ZT12 is lights-off) at 25 °C for 5 d and subsequently released into constant darkness (DD, where circadian time [CT]0 is subjective lights-off). They were harvested onto dry ice every 4 h (unless specified otherwise) during the last day of entrainment and for 1–2 d of DD.

Temperature experiments were conducted entirely in the dark except for the initial seeding of parental bottles. Adults (parental) were placed in fresh media and allowed to mate and lay eggs at 25 °C for 5 d. The parents were cleared and the next generation was kept at 25 °C until early pupal stages. Next, they were transferred to a temperature cycle of 12 h of 18 °C followed by 12 h of 25 °C (CA, where CA0 is onset of 18 °C and CA12 is onset of 25 °C) until eclosion occurred. The newly eclosed flies were transferred to fresh media and allowed to entrain for an additional 4 d and then released into a constant temperature of 25 °C (AA). They were harvested onto dry ice every 4 h during the last day of entrainment and for one to two and a half days in AA (as specified: Day 1 = AA2–22, Day 2 = AA26–46, partial Day 3 = AA50–58). The frozen flies were vortexed and passed through a series of sieves in order to isolate the heads for RNA or protein extraction.

Behavioral analysis.

Individual flies were monitored and their locomotor activity was analyzed with the Drosophila Activity Monitoring System IV (TriKinetics, http://www.trikinetics.com). For LD/DD experiments, the flies were reared under standard lighting conditions and monitored at 25 °C in LD and/or DD as specified. Temperature experiments were conducted entirely in the dark (unless otherwise specified). The flies were raised at 25 °C until the pupal stage and were then entrained to CA until eclosion. Individual flies were monitored and their locomotor activity analyzed as above both during entrainment (CA) and free-run (AA). Period lengths were calculated using ClockLab Software (ActiMetrics, http://www.actimetrics.com).

Northern blot analysis.

Total RNA was extracted from approximately 100 μl of adult heads per time point using either RNA-STAT60 (Tel-Test, Incorporated, http://www.tel-test.com) or guanidinium thiocyanate followed by centrifugation in Cesium chloride solution. 15–30 μg of total RNA was denatured for 5 min at 65 °C and resolved on a 1% formaldehyde-agarose gel (20 mM MOPS [pH7], 5 mM NaOAc, 1 mM EDTA). The resolved RNA was transferred to Nytran membrane (Schleicher & Schuell, http://www.arraying.com) in 10x SSC overnight. Probe templates were radiolabeled as specified for the DECAprime II kit (Ambion, http://www.ambion.com). Hybridizations were carried out at 55 °C in UltraHyb solution (Ambion) supplemented with denatured fish sperm DNA (Roche, http://www.roche.com). Radioactive signals on the blots were visualized and quantitated with either a Storm or Typhoon Phosphorimager (Molecular Dynamics) and the results plotted in Microsoft Excel (http://www.microsoft.com). Fourier analyses were also performed on the northern data as indicated.

Western blot analysis.

Total protein was extracted from about 35 μl of adult heads per time point in 75 μl Head Extraction Buffer (100 mM KCl, 20 mM Hepes [pH 7.5], 10% glycerol, 10 mM EDTA [pH 8], 0.1% Triton X-10, 50 mM NaF, 1 mM DTT) with 1x protease and phosphatase inhibitors (Roche) using a handheld homogenizer (Kontes, http://www.kontes.com). Samples were centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was transferred to a new tube and centrifuged as above for an additional 10 min. 15–30 μg of total protein were resolved on 6% SDS polyacrylamide gels and transferred to nitrocellulose membrane (Schleicher & Schuell). Membranes were blocked for at least 1 h at room temperature with 5% nonfat dry milk in 1x TBST. Primary antibodies were diluted in blocking solution (1:10,000 for α-PER [rabbit], 1:2,000 for α-TIM [rat]) and incubated with the membranes at 4 °C overnight. The membranes were washed three times for 10 min each in 1xTBST and incubated with secondary antibodies (1:10,000) (Jackson ImmunoResearch, http://www.jacksonimmuno.com) for 1 h at room temperature. The membranes were washed as before and detection was carried out using ECL (Amersham Pharmacia Biotech, http://www.gehealthcare.com).

Reverse transcriptase-PCR analysis.

RNA was extracted with RNA-STAT60 (Tel-Test, Incorporated) in the same manner as for northern blots. cDNA was generated using the ThermoScript RT-PCR System (Invitrogen, http://www.invitrogen.com) as described by the manufacturer with one exception: cDNA synthesis was carried out at 50 °C for 90 min with Oligo(dT)20. cDNA (3 μl)was used in subsequent PCR reactions with AccuPrime Pfx DNA Polymerase (Invitrogen).

Microarray experiments.

Two sets of y w flies and one set of arrhythmic mutant y w; tim01 flines were collected as described above for 1 d in CA and 1 d in AA. An additional set of cn bw flies was collected for the first 2 d in AA. RNA was extracted from adult heads with guanidinium thiocyanate followed by centrifugation in Cesium chloride solution. 50 μg of the RNA was further purified over RNeasy columns (Qiagen, http://www1.qiagen.com) according to instructions from the manufacturer. 25 μg of the purified RNA was used to generate biotin-labeled cRNA probe as described in the Affymetrix GeneChip manual. T7-d24 primers (MWG Biotech, http://www.mwg-biotech.com), Superscript Choice (Life Technologies, Invitrogen), and enzymes from New England Biolabs (http://www.neb.com) were used to synthesize cDNA. The ENZO Bioarray High Yield RNA transcript labeling kit was used for in vitro transcription reactions. Hybridization, washing, staining, and scanning of the target cRNA to the Affymetrix Drosophila Genome 1 arrays were carried out according to the Affymetrix GeneChip manual.

Microarray data analysis.

The robust multi-array average (RMA) single algorithm [61] was used to prepare microarray data from each experiment. Using Fourier analysis, 24-h spectral power (F24) was calculated for appended time course experiments as in [27]. To estimate probability values and FDR, a permutated background model was used, in which time ordering of the real data was permuted 1,000 times to give a background distribution of F24. These were divided into a number of quantiles equal to the original number of probe sets and compared to F24 from the real data. Two types of probability values were calculated in association with F24: local p-values assigned to each probe set represent the odds of observing a F24 score of equal or higher strength after random permutation of the time order for the data series of that probe set, whereas global p-values represent the odds of observing an equal or higher F24 in the distribution calculated after random permutation of the time order for the data series of all of the probe sets. The local p-values are used in the selections performed for Figures 3, 4, and 5, whereas the global p-values are used to describe the 24-h periodic expression programs as represented in the graphs of Figures 2 and 6. For the analyses in Figures 2 and 6, only probe sets for which more than half of the values exceeded a 20th percentile cut-off were considered. In Figure 2B–2E, the number of selected rhythmic transcript profiles is plotted as a function of the threshold that is applied to the global p-value. The FDR in Figure 2A simply represents the ratio of the number of transcript profiles selected from the randomly permuted dataset over the number of transcript profiles selected from the real data. The LD data used in Figures 2 and 6 represent the y w (1) and y w (3) time course datasets described in references [27,28], whereas the DD data in Figure 2 represent these time courses plus 2 d of data from [29] formatted as described previously in [27]; the DD₁ dataset in Figure 6 is identical to the DD data in Figure 2, whereas the DD₂ dataset consists of the cn bw time course described in [28] plus 2 d of data from [62] and the average for the day of data from [63]. To select the 164 temperature-driven transcripts illustrated in Figure 3 and Table S1, the local p-value for the CA (2x wt + 1x tim⁰¹) data had to be <0.05, the average absolute daily range of RMA expression values had to be >0.3 for both the CA (2x wt) and the CA (2x wt + 1x tim⁰¹) data and the p-value found for a Kruskal-Wallis test of significant variation with daily time across the CA (2x wt + 1x tim⁰¹) data had to be <0.025. To select the 143 temperature-entrained circadian transcripts illustrated in Figure 4 and Table S1, the local p-value for the CA/AA (2x/4x wt) data had to be <0.001, the average absolute daily range of RMA expression values had to be >0.3, and the p-value found for a Kruskal-Wallis test of significant variation with daily time across the CA/AA (2x/4x wt) data had to be <0.01, and, to avoid light-driven effects, the local p-value for the AA (4x wt) data had to be <0.05. The set of core circadian transcripts in Figure 5 and Table S1 was defined by the overlap of the 143 temperature-entrained circadian transcripts and a set of 172 light-entrained circadian transcripts that was defined based on the following criteria: local p-value for LD/DD (8x/9x wt) had to be <0.001 [27], the average absolute daily range of RMA expression values had to be >0.3, and the p-value found for a Kruskal-Wallis test of significant variation with daily time across the LD/DD (8x/9x wt) data had to be <0.05, and, to avoid light-driven effects, the local p-value for the DD (9x wt) data had to be <0.05. Analysis results are made available at http://biorhythm.rockefeller.edu.

Accession Numbers

The microarray data generated for this study have been deposited under accession number GSE6542 in the Gene Expression Omnibus data repository (http://www.ncbi.nlm.nih.gov/geo). FlyBase (http://www.flybase.net) accession numbers for the Drosophila genes discussed in this paper are Clk (FBgn0023076), cry (FBgn0025680), Pdf (FBgn0023178), Pdp1 (FBgn0016694), per (FBgn0003068), tim (FBgn0014396), and vri (FBgn0016076).