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Proc Natl Acad Sci U S A. Jun 17, 2008; 105(24): 8446–8451.
Published online Jun 6, 2008. doi:  10.1073/pnas.0800145105
PMCID: PMC2448856

Peripheral circadian clock for the cuticle deposition rhythm in Drosophila melanogaster


Insect endocuticle thickens after adult emergence by daily alternating deposition of two chitin layers with different orientation. Although the cuticle deposition rhythm is known to be controlled by a circadian clock in many insects, the site of the driving clock, the photoreceptor for entrainment, and the oscillatory mechanism remain elusive. Here, we show that the cuticle deposition rhythm is regulated by a peripheral oscillator in the epidermis in Drosophila melanogaster. Free-running and entrainment experiments in vitro reveal that the oscillator for the cuticle deposition rhythm is independent of the central clock in the brain driving the locomotor rhythms. The cuticle deposition rhythm is absent in null and dominant-negative mutants of clock genes (i.e., period, timeless, cycle, and Clock), indicating that this oscillator is composed of the same clock genes as the central clock. Entrainment experiments with monochromatic light–dark cycles and cryb flies reveal that a blue light-absorbing photoreceptor, cryptochrome (CRY), acts as a photoreceptor pigment for the entrainment of the cuticle deposition rhythm. Unlike other peripheral rhythms in D. melanogaster, the cuticle deposition rhythm persisted in cryb and cryOUT mutant flies, indicating that CRY does not play a core role in the rhythm generation in the epidermal oscillator.

Keywords: circadian rhythm, clock genes, cryptochrome, entrainment, epidermal cell

Circadian clocks control daily rhythms at the molecular, physiological, and behavioral levels. Like most organisms, the central circadian oscillator controlling the locomotor activity rhythm in Drosophila is proposed to consist of molecular feedback loops. The feedback loops comprise several core clock genes, such as period (per), timeless (tim), Clock (Clk), and cycle (cyc) (1). These genes encode PER, TIM, CLK, and CYC proteins, respectively, and loss-of-function or dominant-negative mutations in these genes result in loss of normal circadian rhythmicity (25). CLK and CYC form a heterodimer to activate transcription of per and tim (2, 4, 6). PER and TIM form another heterodimer, are transferred into the nucleus, and then inactivate the transcriptional activity of the CLK/CYC heterodimer (6). The feedback loops are entrained and reset by the light-induced degradation of TIM mediated by a photoreceptor pigment, cryptochrome (CRY) (710).

In addition to the central oscillator, peripheral oscillators exist in many tissues, including the compound eyes, antennae, wings, legs, and Malpighian tubules in adults and the ring gland in pupae (1114). These oscillators are self-sustained and directly light-entrainable even in vitro (1113). In addition, the oscillators are clearly independent of the central circadian oscillator(s) in the brain (15, 16). Previous reports revealed that the peripheral oscillator shares the same genes with the central clock in the brain (12, 17). However, there is a remarkable difference that, in the peripheral oscillator, CRY functions as a core component besides a photoreceptor. In cryb (a cry loss-of-function mutant) and cry0 (a KO mutant of cry) flies, molecular oscillations of the clock genes and proteins are absent in peripheral tissues (i.e., the compound eyes, antennae, legs, and Malpighian tubules) (10, 1821). In the compound eyes, CRY is suggested to act as a repressor of the CLK/CYC transcriptional activity (22).

Insect exoskeleton, consisting of epi-, exo-, and endocuticle, thickens by additional deposition of the endocuticle materials after adult emergence. They are secreted by epidermal cells in layers in which the chitin microfibrils exist in different orientations, unidirection and helicoid (23). Such cuticle layers in the endocuticle are well known in a wide range of insects and are observed as dark-bright bands in the endocuticle under a Nomarski differential interference contrast microscope or polarization microscope (24). It has been reported that the cuticle layers increase at the rate of one per day, and the rhythm persists even in vitro in cockroaches (2527), suggesting that the rhythm is governed by a peripheral oscillator. However, the molecular mechanisms for the cuticle deposition rhythm, including oscillation and entrainment, have not been documented.

Here, we clarified in D. melanogaster that the cuticle deposition rhythm at the furca, thoracic apodeme attached to inner muscles, is controlled by a peripheral circadian oscillator localized in the epidermis and the oscillator involves per, tim, cyc, and Clk. Furthermore, we suggest the involvement of the per- and tim-independent oscillators because per01 and tim01 flies showed rhythmic cuticle deposition only under light–dark (LD) cycles of 24 h, but not under LD cycles of 21 or 28 h. Photic entrainment in the cuticle deposition rhythm occurred even when isolated thoraxes were cultured in vitro, indicating that the photoreception site resides in peripheral tissues. In contrast to the other peripheral oscillators, cryb flies showed a clear cuticle deposition rhythm without photic entrainment. The entrainability was restored by rescue of cry function. From these results, we conclude that CRY is not a core component, but an exclusive photoreceptor in the cuticle deposition rhythm.


Regulation of Cuticle Deposition by a Circadian Oscillator.

In Drosophila, the cuticle growth layers are observed only in the phragma, the second and third furcae in the thorax (28). They are the apodemata (i.e., internal cuticular processes of the body wall on which the muscles are inserted). We used the third furca for observation because it was easiest to be dissected out, and a layer in the other apodemata often was difficult to be discerned in flies older than 5 days (28). To characterize the cuticle deposition rhythm, we first examined the cuticle deposition under various light and temperature conditions in WT flies. Alternating bright and dark layers were observed in the endocuticle (Fig. 1A Left), with the number of bright layers increasing at the rate of about one per day, with little variations until day 7 under LD cycles with the Zeitgeber period (T) = 24 h at 25°C. After day 7, the increase rate of cuticle layers declined slightly (Fig. 1B). This cuticle deposition rhythm persisted under constant darkness (DD) (Fig. 1B). Hereafter, we compared the numbers of layers on day 6 in flies kept under various conditions because of the decline of the increase rate after day 7.

Fig. 1.
Circadian properties of the cuticle deposition rhythm in adult D. melanogaster. (A) Sagittal view of the endocuticle in the third furca on day 6. Alternating layers are observed in WT flies kept under LD cycles of T = 24 h (Left), but not in the per01 ...

The number of layers (mean ± SD) on day 6 under DD at 22.5, 25, and 27.5°C was 7.06 ± 0.83 (n = 33), 7.22 ± 0.83 (n = 36), and 6.94 ± 0.72 (n = 32), respectively, with no significant difference among the three temperatures (ANOVA, P > 0.05). The Q10 value of the oscillation rate calculated from the means was 1.09 at 22.5–25°C and 0.85 at 25–27.5°C. Thus, the free-running period of the rhythm is temperature-compensated.

We next examined the entrainability of the cuticle deposition rhythm in WT flies to LD cycles with various periods (T) for 6 days. If the rhythm is entrained to LD cycles, the number of layers would change as shown by the hyperbola in Fig. 1C (y = 1 + 144/x) in accordance with the number of LD cycles. When T = 21–28 h, all flies showed bright and dark layers in the endocuticle, and the mean number of bright layers was changed along the hyperbola with a little variance (Fig. 1C). The number of layers in T = 21 h was significantly larger than those in T = 24 h and 28 h (Tukey test, P < 0.01). Although the mean number of layers was significantly higher than the hypothesized value in T = 28 h (t test, P < 0.01), its variance was small, and, therefore, the rhythm seemed to entrain to the LD cycles in most individuals. On the contrary, when T = 16–18 and 36–48 h, the number of bright layers did not change along the hyperbola and was significantly different from the hypothesized values (t test, P < 0.001). In addition, the variance in the number of bright layers was larger than that in 21, 24, and 28 h (Tukey-type multiple comparison for variations, P < 0.01) (29). These results indicate that the cuticle deposition rhythm is entrained to LD cycles within the range of T = 21–28 h. Hereafter, we considered that the cuticle deposition rhythm is entrained to LD cycles when the numbers of bright layers are changed with the same pattern as that of WT flies under LD cycles with T = 21–28 h (see Fig. 1C).

Thus, the cuticle deposition rhythm showed the three major properties of the circadian rhythm: persistence of an overt rhythm under constant conditions, temperature compensation of its free-running period, and stable entrainment to environmental cues with periods in a limited range ≈24 h (30). We conclude, therefore, that the rhythm is controlled by a circadian clock.

The variance in the number of bright layers was significantly larger under LD cycles with T = 16, 18, and 48 h than under DD (F test, P < 0.05) (Fig. 1C), although the mean number was not significantly different among LD cycles with T = 16, 18, and 48 h and DD (Aspin–Welch t test, P > 0.05), suggesting that the cuticle deposition rhythm did not simply free-run in some individuals outside the entrainment range. In addition, some fraction of flies did not show bright and dark layers under LD cycles with T = 16, 18, and 36 h (8.6, 17.3, and 8.3% of individuals, respectively), but all of the individuals showed alternating bright and dark layers under LD cycles with T = 48 h. These results indicate that the cuticle deposition rhythm in some individuals is unstable under extreme LD regimes except T = 48 h. We suggest that the cuticle deposition rhythm free-runs with frequency demultiplication and relative coordination outside the entrainment range, as reported in various circadian rhythms (30, 31).

Peripheral Clock for the Cuticle Deposition Rhythm.

To examine whether the cuticle deposition rhythm is independent of the central clock(s) in the brain, we tested the cuticle deposition rhythm in isolated whole thoraxes or furcae cultured in vitro from day 1. Seven bright layers were formed in the endocuticle of most thoraxes and furcae cultured under DD on day 6 (Fig. 2A), indicating that the rhythm is generated by a clock within the isolated tissues. It is noteworthy that the cuticle deposition rhythm was entrained to LD cycles with T = 21–28 h in thoraxes cultured in vitro (Fig. 2A Left), but not in isolated furcae (Fig. 2A Right).

Fig. 2.
Localization of the circadian oscillator for the cuticle deposition rhythm in WT D. melanogaster. (A) Persistence of the cuticle deposition rhythm under DD and its entrainment to LD cycles in vitro. Isolated thoraxes (Left) and furcae (Right) were cultured ...

We then confirmed the presence of a peripheral clock for the cuticle deposition rhythm in the furcae by immunohistochemistry using PER antiserum. PER immunoreactivity was found in the nuclei of epidermal cells in the outermost part of an apodeme in 26 of 29 specimens at Zeitgeber time (ZT) 23 (Fig. 2B). No immunoreactivity, however, was observed in all nine specimens at ZT 5. Immunoreactivity was observed in 3 of 23 specimens at ZT 8 and 5 of 12 specimens at ZT 11. These results suggest that the epidermal cells include the oscillator controlling the cuticle rhythm.

Cuticle Deposition Rhythm in Clock Mutant Flies.

To examine the involvement of clock genes in the cuticle deposition rhythm, we tested the deposition of the endocuticle in per01, tim01, cyc01, ClkJrk, and cryb mutants under LD cycles with T = 21–28 h and DD. In WT and cryb flies, bright and dark layers in the endocuticle were observed in all individuals under all of the conditions examined (Fig. 3). In most of the per01 flies, the endocuticle was thickened without distinct layers under DD (Figs. 1A Right and and3).3). Most of the tim01, cyc01, and ClkJrk flies also showed similar patterns under DD (Fig. 3). Therefore, the circadian clock genes are involved in the timing of change in the chitin arrangement, and a loss of the cuticle deposition rhythm is expressed as an absence of alternating layers in the endocuticle.

Fig. 3.
Effects of clock gene mutations on the cuticle deposition rhythm in D. melanogaster. Flies were kept under DD or LD cycles with T = 21–28 h. The ordinate shows the percentage of the individuals with alternating layers in the endocuticle on day ...

The results under LD cycles were different between per01, tim01 flies and cyc01, ClkJrk flies. No alternating layers were shown by 50–80% cyc01 and ClkJrk flies in the endocuticle in all test conditions, indicating that cyc and Clk are crucial components for the cuticle deposition rhythm (Fig. 3). Most of the per01 and tim01 flies, in contrast, showed alternating layers when T was 24 h (Fig. 3). If the formation of alternating layers with T = 24 h is a direct response to light, it should be observed even when T is different from 24 h. However, most per01 and tim01 flies showed no bright and dark layers under LD cycles with T = 21 and 28 h. Therefore, the formation of cuticle layers also can be achieved by a per- or tim-independent circadian clock. This clock depends on cyc and Clk and oscillates only under LD cycles with T close to 24 h.

Because CRY is a photoreceptor for entrainment of the cuticle deposition rhythm (see next section), we examined the feature of per-independent rhythm by using per01; cryb flies. More than 90% (33 of 35) of per01; cryb flies tested had no alternating layers in the endocuticle even when T was 24 h, suggesting that per-independent oscillation needs light input via CRY.

Role of CRY in the Cuticle Deposition Rhythm.

To search for the photopigment for entrainment of the cuticle deposition rhythm, we examined the range of effective wavelengths in WT flies using monochromatic LD cycles with T = 21–28 h. The rhythm was found to be entrained to LD cycles of blue (470 nm), but not to LD cycles of yellow (583 nm) and red (660 nm) (Fig. 4). These results indicate that entrainment of the cuticle deposition rhythm is achieved by a blue light-absorbing photopigment. We assume that CRY is the pigment because CRY is a blue light-absorbing photopigment to reset and entrain the circadian locomotor rhythm in D. melanogaster (1).

Fig. 4.
The effect of monochromatic LD cycles on the entrainment of the cuticle deposition rhythm in WT D. melanogaster. The illumination of blue (wavelength = 470 nm), yellow (583 nm), and red (660 nm) was used instead of the white fluorescent light from the ...

To clarify the role of CRY, we examined the cuticle deposition rhythm in cryb. The rhythmic deposition of the endocuticle was clearly formed in most individuals, and the number of bright layers increased at the rate of about one per day under both LD cycles with T = 24 h and DD (Fig. 5A). Although cryb is a loss-of-function mutant of cry, it leaks out a low level of its encoded protein (32). To exclude the possibility that leaking proteins participate in driving the oscillation for the cuticle deposition rhythm in cryb, we tested the cuticle deposition under DD in another cry mutant, cryOUT, in which no CRY protein is detected by immunohistochemistry (33). Bright and dark layers in the endocuticle were observed in all cryOUT flies, and the number of bright layers on day 6 was 7.13 ± 1.34 (mean ± SD, n = 41), suggesting that circadian cuticle deposition rhythm free-ran in cryOUT. The persistence of the rhythm in cryb and cryOUT under DD shows that, unlike other peripheral circadian rhythms (9, 10, 1821), cry seems not the core component for the generation of the cuticle deposition rhythm.

Fig. 5.
Circadian properties of the cuticle deposition in cryb adults of D. melanogaster. (A) The daily increase of growth layers of the endocuticle under LD cycles of T = 24 h (open circles) and DD (filled circles) (the dotted line: see 1B). Alternating layers ...

The cuticle deposition rhythm in cryb flies was not entrained to LD cycles with T = 21–28 h (Fig. 5B). We rescued cryb by act-Gal4-mediated cry expression. Control flies having either a UAS-cry responder or an act-Gal4 driver in cryb background were not entrained to LD cycles like cryb flies (Fig. 6). In act-Gal4/UAS-cry; cryb flies, however, the cuticle deposition rhythm is entrained to LD cycles with T = 21–28 h (Fig. 6), and the range of entrainment was the same as that in the WT flies (see Fig. 1C). These results show the involvement of cry in photic entrainment of the cuticle deposition rhythm.

Fig. 6.
Restoration of entrainment ability of the cuticle deposition rhythm to LD cycles in cryb flies by act-Gal4-mediated cry expression. The number of bright layers on day 6 is shown (hyperbola: see Fig. 1C). Entrainment to LD cycles was examined in cryb flies ...


Peripheral Circadian System for Cuticle Deposition Rhythm.

The cuticle deposition rhythm is crucial for producing the correct alternating layers with different chitin orientations, which increases the physical strength of the cuticle (23). The present results demonstrated that this rhythm is achieved by the circadian clock residing in the epidermal cells. PER signals were detected solidly in the nuclei late at night and weakly during the day as reported for the cerebral pacemaker neurons, photoreceptor cells of the compound eyes, and the Malpighian tubules of D. melanogaster (3437). The peripheral oscillator controlling the cuticle deposition rhythm seems independent of the central clock in the brain, such as that in the antennae and Malpighian tubules (15, 16), because the ranges of the Zeitgeber period and effective wavelengths for entrainment were different from the locomotor rhythm; both ranges were distinctively narrower than those in the circadian locomotor rhythm in D. melanogaster (3841). To investigate whether the rhythm is completely independent of the central clock, transplantation experiments between individuals with different phasing or experiments of specific ablation of central pacemaker cells are necessary. Similar endocuticle deposition rhythm has been reported in cockroaches and locusts: The cuticle deposition rhythm persists under DD with a temperature-compensated free-running period of ≈24 h (27, 42). The rhythm is neither entrained to LD cycles nor reset by light in cockroaches (27) but coincides with the period of LD cycles such as T = 12 and 48 h in locusts (43). In locusts, it is unclear whether the coincidence of the rhythm with LD cycles is indeed entrainment of the clock or a direct response to LD cycles. In contrast, the cuticle deposition rhythm in D. melanogaster is shown to be driven by a system consisting of the oscillator and photoreceptors, thus suitable for a study on the entrainment mechanism of a circadian peripheral oscillator.

Oscillator Mechanism for the Cuticle Deposition Rhythm.

The circadian clock for the cuticle deposition rhythm requires the clock genes per, tim, cyc, and Clk because no alternating bright and dark layers were formed in per01, tim01, cyc01, and ClkJrk mutant flies in DD. This finding indicates that the epidermal oscillator shares the same clock genes with the central oscillator of locomotor activity rhythm (25) and also would be composed of the molecular feedback loops reported for central circadian clock(s) (1).

Surprisingly, alternating cuticle layers were observed in the cryb mutant flies in all conditions examined and in cryOUT flies under DD, indicating that CRY is not involved in oscillation for cuticle deposition rhythm. This result is in contrast to other peripheral tissues, including the antennae, legs, photoreceptor cells, and Malpighian tubules, where CRY plays a role as a core component of the circadian oscillator and the circadian molecular oscillations are absent in cryb or cry0 flies in DD (9, 10, 1821). In the photoreceptor cells of the compound eyes, CRY functions as a transcriptional repressor of the CLK-CYC-activated transcription (22). This finding explains why circadian clocks in peripheral tissues stop in cryb and cry0 mutants. Because the cuticle deposition rhythm sustains in cryb mutants, the molecular mechanism underlying the endocuticular oscillator seems to be different from other peripheral clocks but resembles the cerebral central clocks.

Interestingly, cuticle deposition of per01 and tim01 was rhythmic in LD cycles with T = 24 h. The rhythm is not a direct response to light because the rhythm never persisted in DD and LD cycles with T = 21 and 28 h. The result indicates that there is a per- or tim-independent circadian oscillator. On the contrary, cuticle deposition of ClkJrk and cyc01 was arrhythmic in both DD and all LD cycles examined. Therefore, Clk and cyc must be essential components for the per- or tim-independent circadian oscillator. The oscillator seems to have a narrow range of entrainment, close to 24 h, and seems to be quickly damped under DD because per01 and tim01 flies produced no alternating layers under DD. The photic input mediated by CRY is required for the per- independent oscillation because the cuticle deposition rhythm was absent in the double mutant, per01; cryb, even under LD cycles with T = 24 h. The involvement of a per- or tim-independent circadian oscillation also has been reported in locomotor rhythms and in the fluorescent dye incorporation rhythm of the salivary gland (38, 39, 44, 45). Under LD cycles with various T, per0 flies show a limited entrainment range, and entrained flies exhibit a characteristic phase relationship of the locomotor activity rhythm to LD cycles, indicating that per0 still contains a circadian oscillator system (39). Furthermore, analysis of this secondary oscillator using temperature cycles with different T reveals the existence of the per- or tim-independent oscillator that contains Clk and cyc as indispensable components in the central clock (44). The present results also support this idea in the peripheral clocks controlling the cuticle deposition rhythm. Although little is known about the per-independent oscillation in peripheral tissues, the secondary oscillator of the cuticle deposition rhythm driven by CRY-dependent light input would serve as a model to reveal its mechanism.

Entrainment System for Cuticle Deposition Rhythm.

The cuticle deposition rhythm in the furca was entrained to LD cycles when the whole isolated thorax was cultured in vitro, but not when the furca separated from the other parts of the thorax was cultured. These results suggest that the primary photoreceptor is present in the thorax and that the known external circadian photoreceptors, such as the compound eyes, ocelli, and Hofbauer–Buchner's eyelets (9, 10, 4648), do not play a primary role in photic entrainment. Although the circadian oscillator driving the cuticle deposition rhythm is most likely to be present in the epidermal cells, the precise site of photoreception for entrainment in the thorax remains unclear. One possible explanation is that the photoreceptor is in the epidermal cells, but the entrainment system may not be operative under our in vitro conditions. It seems more probable, however, that the photoreceptor is in some tissue other than the epidermal cells in the thorax. Identification of the extraretinal receptor in the thorax is an interesting next issue. In this case, a humoral factor might be involved in the entrainment. This possibility would be examined by transplantation of the furca to the abdomen of another fly to see whether the transplanted furca entrains to LD cycles. The entrainment experiments with monochromatic light and using cryb flies revealed that CRY is the essential photoreceptor for the entrainment of the cuticle deposition rhythm. The rescue of photic entrainability in cryb flies by act-Gal4-mediated cry expression also supports this view. Therefore, our results provide evidence that CRY is a predominant photoreceptor for the entrainment in the cuticle deposition rhythm. Thus, the circadian system for the cuticle deposition rhythm is similar to the central clock in the brain, which differs from the other peripheral oscillators.

Materials and Methods

Fly Strains' Rearing and Collection.

Flies were reared on standard medium containing wheat germ at 25°C under 12-h LD cycles (LD 12:12, 10:00–22:00 light). Newly emerged flies collected within 6 h from light-on were used in all experiments. Day 0 is defined as 24 h from light-on of the day of adult emergence, and the next 24 h is day 1. Canton-S was used as WT. The following mutants and transgenic strains were used: per01, tim01, cyc01, ClkJrk, cryb, cryOUT (25, 10, 33), per01; cryb, act-Gal4/UAS-cry; cryb. per01; cryb flies were generated by per01 and cryb ss1 (aka rec6). act-Gal4/UAS-cry; cryb flies were obtained by crossing act-Gal4/CyO; cryb and UAS-cry; cryb (49). All experiments were performed at 25°C, with exceptional cases described separately. The white fluorescent light (FLR20 SW/M or FL15W; Matsushita Electric Works) was used at an intensity of 3 W/m2 for the photophase in all experiments.

Histological Observation of Cuticle Growth Layers.

Flies exposed to various conditions were fixed in a solution of 3:1 ethanol and acetic acid for 24 h. Isolated thoraxes were boiled in 4% sodium hydroxide solution to remove the organs and proteins. Cleaned thoraxes were stained with 1% KMnO4. Finally, the third furcae were dissected and mounted in glycerol on a slide glass. Growth layers were observed at 400× and 600× under a Nomarski differential interference contrast microscope (BX50-DIG; Olympus).

Daily Increase of Cuticle Growth Layers.

Flies were exposed to LD 12:12 and DD (DD started from 22:00 on the day of adult emergence) and were fixed in the photophase and subjective day, respectively, on each day. A distinct eclosion line marked the extent of a preeclosion furca at the end and side of a furca. This line was bright under a differential interference contrast microscope and was termed an “eclosion layer” (28). In the present study, the eclosion layer also was counted as a bright layer.

Effect of Temperature on Cuticle Growth Layers.

Flies reared at 22.5 or 27.5°C under LD 12:12 from eggs were kept under DD (DD started from 22:00 on the day of adult emergence) at the same temperature. Flies were fixed in the subjective day on day 6.

Effect of LD Cycles on Cuticle Deposition Rhythm.

Flies were exposed to LD cycles with T = 16, 18, 21, 24, 28, 36, or 48 h consisting of equal durations of the photophase and scotophase. In these regimes, 9, 8, 7, 6, 5, 4, or 3 LD cycles were given to flies during 6 days (144 h). Flies were fixed in the photophase on day 6.

Effect of Monochromatic LD Cycles on Entrainment.

White fluorescent light was used in the first photophase, and monochromatic light was applied from the second photophase. Flies were exposed to monochromatic LD cycles and fixed in the photophase on day 6. The illumination of the monochromatic light was produced with blue LED (SLP-0137A-51, λmax = 470 nm; Sanyo Electric), yellow LED (TLOH180P, λmax = 583 nm; Toshiba), and red LED (SLP-838A-37S1, λmax = 660 nm; Sanyo Electric). The light intensity was adjusted to 2.2–2.9 × 1023 photons per s·cm2 for 470, 583, and 660 nm with a radiometer/photometer (UDP 161; United Detector Technology).

Tissue Culture.

Flies were exposed to one LD cycle (T = 21–28 h), and thoraxes or furcae were dissected on the second photophase. They were cultured individually in 150 μl of Schneider's medium (Invitrogen) including 0.1% penicillin-streptomycin (Invitrogen) under LD cycles for 5 days. Cultured thoraxes or furcae were fixed in the photophase. In addition, tissue culture was performed under DD. In this case, thoraxes or furcae were isolated on the second photophase of LD 12:12, cultured in DD (DD started from the second scotophase) for 5 days, and fixed in the subjective day on day 6.

Immunohistochemistry and Nuclear Staining of Furcae.

WT flies reared under LD 12:12 were fixed at ZT 5, 8, 11, and 23 on day 6 by submersion in 4% paraformaldehyde in phosphate buffer (pH 7.3) with 0.1% Triton X-100. The third furcae were dissected as whole mounts. The primary and secondary antibodies used were 1:2,000 anti-PER antiserum (Santa Cruz Biotechnology) and 1:200 biotinylated anti-goat secondary antibody (Jackson ImmunoResearch), respectively. Signals were enhanced by tyramide-biotin system (PerkinElmer). Visualization was done with streptavidin-Alexa Fluor 488 (Molecular Probes). Some immunostained furcae were counterstained by propidium iodide. Preparations were imaged by laser-scanning confocal microscopy (FLUOVIEW; Olympus). Digital images were processed by using Adobe Photoshop 6.0 (Adobe Systems) and Corel Draw 11.0 (Corel).


We thank J. C. Hall (Brandies University, Waltham, MA) for cryb ss1 (aka rec6), A. Matsumoto (Kyushu University, Fukuoka, Japan) for UAS-cry; cryb flies, R. Stanewsky (Queen Mary University of London, United Kingdom) for cryOUT flies, and S. Tamotsu for permitting us to use the radiometer/photometer and helping in measuring the light intensity.


The authors declare no conflict of interest.

This article is a PNAS Direct Submission.


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