A new rhythm mutation was isolated based on its elimination of per-controlled luciferase cycling. Levels of period or timeless clock gene products in the mutant are flat in daily light-dark cycles or constant darkness (although PER and TIM oscillate normally in temperature cycles). Consistent with the fact that light normally suppresses TIM, cryb is an apparent null mutation in a gene encoding Drosophila's version of the blue light receptor cryptochrome. Behaviorally, cryb exhibits poor synchronization to light-dark cycles in genetic backgrounds that cause external blindness or demand several hours of daily rhythm resets, and it shows no response to brief light pulses. cryb flies are rhythmic in constant darkness, correlating with robust PER and TIM cycling in certain pacemaker neurons.
Circadian rhythms are entrained by light to follow the daily solar cycle. We show that Drosophila uses at least three light input pathways for this entrainment: (1) cryptochrome, acting in the pacemaker cells themselves, (2) the compound eyes, and (3) extraocular photoreception, possibly involving an internal structure known as the Hofbauer-Buchner eyelet, which is located underneath the compound eye and projects to the pacemaker center in the brain. Although influencing the circadian system in different ways, each input pathway appears capable of entraining circadian rhythms at the molecular and behavioral level. This entrainment is completely abolished in glass(60j) cry(b) double mutants, which lack all known external and internal eye structures in addition to being devoid of cryptochrome.
cry (cryptochrome) is an important clock gene, and recent data indicate that it encodes a critical circadian photoreceptor in Drosophila. A mutant allele, cry(b), inhibits circadian photoresponses. Restricting CRY expression to specific fly tissues shows that CRY expression is needed in a cell-autonomous fashion for oscillators present in different locations. CRY overexpression in brain pacemaker cells increases behavioral photosensitivity, and this restricted CRY expression also rescues all circadian defects of cry(b) behavior. As wild-type pacemaker neurons express CRY, the results indicate that they make a striking contribution to all aspects of behavioral circadian rhythms and are directly light responsive. These brain neurons therefore contain an identified deep brain photoreceptor, as well as the other circadian elements: a central pace-maker and a behavioral output system.
To determine the in vivo regulatory pattern of the clock gene period (per), the authors recently developed transgenic Drosophila carrying a luciferase cDNA fused to the promoter region of per. They have now carried out noninvasive, high time-resolution experiments allowing high-throughput monitoring of circadian bioluminescence rhythms in individual living adults for several days. This immediately solved several problems (resulting directly from individual asynchrony within a population) that have accompanied previous biochemical experiments in which groups of animals were sacrificed at each time point. Furthermore, the authors have developed numerical analysis methods for automatically determining rhythmicity associated with bioluminescence records from single flies. This has revealed some features of per gene transcription that were previously unappreciated and provides a general strategy for the analysis of rhythmic time series in the study of molecular rhythms.
The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock of Drosophila melanogaster. PDF is necessary for robust activity rhythms and is thought to couple the circadian oscillations of the clock neurons. However, little is known about the action of PDF on individual clock neurons. Here, we combined the period-luciferase reporter system with immunolabeling of clock proteins in wild-type and Pdf 01 mutants to dissect the effects of PDF on specific subgroups of clock neurons. Additionally, PDF levels were elevated to higher than normal levels using specific neural mutants, and a correlation analysis of locomotor activity and clock protein staining served to determine the periods of specific clock cells. We found that PDF has multiple effects on the clock neurons: In some groups of clock neurons, PDF was required for maintaining the oscillations of individual cells, and in others, PDF was required for synchronous cycling of the individual members. Other clock neurons cycled with high amplitude in absence of PDF, but PDF affected their intrinsic clock speed. Sometimes PDF shortened and sometimes PDF lengthened period. Our observations indicate that PDF is crucial for adjusting cycling amplitude, period, and phase of the different players in the circadian clock. Under natural conditions PDF may be required for adapting Drosophila's clock to varying photoperiods. Indeed, we show here that Pdf 01 mutants are not able to adapt their activity to long photoperiods in a wild-type manner.
We conclude that temperature synchronization of the circadian clock is a tissue-autonomous process that is able to override the arrhythmia-inducing effects of constant light. Our data suggest that it involves a cell-autonomous signal-transduction cascade from a thermal receptor to the circadian clock. This process includes the function of phospholipase C and the product specified by the novel mutation nocte.
Cryptochrome (CRY) is intimately associated with the circadian clock of many organisms. In the fruit fly Drosophila melanogaster, CRY seems to be involved in photoreception as well as in the core clockwork. In spite of the critical role of CRY for the clock of Drosophila, it was not quite clear whether CRY is expressed in every clock cell. With the help of a new antibody and a mutant that lacks CRY, we show here that CRY is expressed in specific subsets of Drosophila's pacemaker neurons and in the photoreceptor cells of the compound eyes. In the pacemaker neurons, CRY levels and kinetics under light-dark cycles are quite different from each other. High-amplitude oscillations are observed in only three groups of clock neurons, suggesting that these three groups are strongly receptive to light. The different CRY kinetics may account for phase differences in oscillations of the clock proteins observed in these three groups in earlier studies. The molecular clock of the neurons that contain lower CRY levels or are completely CRY negative can still be synchronized by light, probably via intercellular communication with the CRY-positive neurons as well as via external photoreceptors.
The fly Drosophila melanogaster possesses five photoreceptors and/or photopigments that appear to be involved in light reception and synchronization of the circadian clock: (1) the compound eyes, (2) the ocelli, (3) the Hofbauer-Buchner eyelets, (4) the blue-light photopigment cryptochrome, and (5) unknown photopigments in the clock-gene-expressing dorsal neurons. To understand the contributions of these photoreceptors and photopigments to synchronization, the authors monitored the flies' activity rhythms under artificial long and short days. They found that all the different photoreceptors and photopigments contribute significantly to entrainment under each photoperiod, but the compound eyes are especially important for entrainment to extreme photoperiods. The compound eyes are, furthermore, necessary for adjusting the phase of the activity rhythm, for distinguishing long days from constant light, and for the normal masking effects of light--namely, promotion of activity by lights-on and inhibition of activity by darkness. Cryptochrome is important for period lengthening under long days, although it is more important for entrainment to short days than to long days and is, furthermore, important for aftereffects of the photoperiod on the internal clock. The specific roles of the remaining photoreceptors are more difficult to assess.
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