Cryptochrome antagonizes synchronization of Drosophila's circadian clock to temperature cycles

School of Biological and Chemical Sciences, Queen Mary,University of London, London E1 4NS, UKSummaryBackground: In nature, both daily light:dark cycles andtemperature fluctuations are used by organisms to synchro-nize their endogenous time with the daily cycles of light andtemperature.Propersynchronization isimportantfortheover-all fitness and wellbeing of animals and humans, and althoughwe know a lot about light synchronization, this is not the casefor temperature inputs to the circadian clock. In Drosophila,light and temperature cues can act as synchronization signals(Zeitgeber), but it is not known how they are integrated.Results: We investigated whether different groups of theDrosophila clock neurons that regulate behavioral rhythmicitycontribute to temperature synchronization at different abso-lute temperatures. Using spatially restricted expression ofthe clock gene period, we show that dorsally located clockneurons mainly mediate synchronization to higher (20 C:29 C) and ventral clock neurons to lower (16 C:25 C) temper-ature cycles. Molecularly, the blue-light photoreceptorCRYPTOCHROME (CRY) dampens temperature-inducedPERIOD (PER)-LUCIFERASE oscillations in dorsal clockneurons. Consistent with this finding, we show that in theabsenceof CRYverylimitedexpression ofPERin afewdorsalclock neurons is able to mediate behavioral temperaturesynchronization to high and low temperature cycles indepen-dent of light.Conclusions:Weshowthatdifferentsubsetsofclockneuronsoperate at high and low temperatures to mediate clocksynchronization to temperature cycles, suggesting thattemperature entrainment is not restricted to measuring theamplitude of such cycles. CRY dampens temperature inputto the clock and thereby contributes to the integration ofdifferent Zeitgebers.IntroductionInnature,organismsuseanarrayoftemporalinformationfromthe environment to synchronize their circadian clocks. InDrosophila,dailylightandtemperaturechangesareperceiveddirectly by either clock cells [1–3] or specialized ‘‘circadian’’photoreceptors [4, 5] and by sensory systems that are knownto function in image forming vision and mechanoreception[5, 6]. The various ‘‘Zeitgebers’’ are thought to be integratedby the pacemaker neurons for an accurate estimate of daytime and optimal timing of behavioral activities (e.g., [7]). Inmammals, the clock neurons in suprachiasmatic nuclei arenot directly sensitive to light and temperature fluctuations.Similar to what is seen in flies, the circadian clock receiveslight information from the image forming photoreceptors, aswell as from specialized melanopsin-expressing retinalganglion cells [8]. The suprachiasmatic nucleus seems to belargely inert to temperature fluctuations, and it is thoughtthat the daily body temperature fluctuations are regulated bythe suprachiasmatic nuclei to synchronize peripheral clocktissues [9–11].In insects, temperature acts as potent Zeitgeber (e.g.,temperature cycles with an amplitude of 2 C–3 C stablysynchronize the Drosophila clock [12, 13]) and may even bethe dominant cue in nature [14]. Also, light and temperaturesynchronizetheclockinterdependently[7,13–16],andinorderto understand Zeitgeber integration it is crucial to revealtemperature entrainment mechanisms as such. Although inperipheral fly tissues temperature synchronization seems tobe a cell-autonomous mechanism [2] at least some of thepacemaker neurons in the brain receive temperature informa-tion from internal mechanoreceptive sensory structureslocated in fly appendages [6]. This and potential other directand indirect temperature inputs result in synchronized clockgene expression within the clock circuitry [7, 17, 18]. Thiscircuitconsistsofabout150neuronsalldefinedbytheexpres-sion of the clock proteins PERIOD (PER) and TIMELESS (TIM),which are divided into several subgroups based on their loca-tion, size, projection pattern, and neuropeptide content [19].The ventral lateral neurons consist of large (l-LNv) and small(s-LNv) neurons, which also express the neuropeptidepigment dispersing factor (PDF), and one PDF-negatives-LNv (the fifth s-LNv). The other groups are the dorsal lateralneurons (LNds), three groups of dorsal neurons (DN1–DN3),and lateral posterior neurons (LPNs). Although all clockneurons show similar phases of PER and TIM expression,some neurons, in particular the LPN and DN2, are thought tobe more sensitive to temperature, because they preferablysynchronize to temperature in conflicting light:dark (LD) andtemperature cycles [18, 20]. Interestingly, these neuronsbelong to the groups that do not contain the circadian photo-receptor CRYPTOCHROME (CRY), leading to the hypothesisthatCRY-negativeneuronsmainlymediatetemperatureinputs[20].Ontheotherhand,fliesthathaveafunctionalclockonlyinCRY-positive neurons are able to synchronize to temperaturecycles, indicating that CRY-positive neurons contribute totemperature entrainment [21]. We and others have shownthat in constant light (LL) temperature cycles induce robustclock gene oscillations in peripheral clock cells and in theclock neurons as well as synchronized behavior [2, 17]. Atconstant temperatures LL leads to molecular and behavioralarrhythmicity, and therefore temperature cycles somehowinterfere with the effects of light on the molecular clock[2, 17, 22–24].Inthecurrentstudy,weaimedtoidentifytheroleofdifferentclock neurons and CRY in temperature entrainment in the