Ralf Stanewsky

The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila.

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 Drosophilas 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.

Quantitative Analysis of Drosophila period Gene Transcription in Living Animals

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 asyn chrony within a population) that have accompanied previous biochemical ex periments in which groups of animals were sacrificed at each time point. Furthermore, the authors have developed numerical analysis methods for auto matically 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.

Drosophila CRY is a deep brain circadian photoreceptor.

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.

The Circadian Clock of Fruit Flies Is Blind after Elimination of All Known Photoreceptors

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.

Temperature Synchronization of the Drosophila Circadian Clock

BACKGROUND Circadian clocks are synchronized by both light:dark cycles and by temperature fluctuations. Although it has long been known that temperature cycles can robustly entrain Drosophila locomotor rhythms, nothing is known about the molecular mechanisms involved. RESULTS We show here that temperature cycles induce synchronized behavioral rhythms and oscillations of the clock proteins PERIOD and TIMELESS in constant light, a situation that normally leads to molecular and behavioral arrhythmicity. We show that expression of the Drosophila clock gene period can be entrained by temperature cycles in cultured body parts and isolated brains. Further, we show that the phospholipase C encoded by the norpA gene contributes to thermal entrainment, suggesting that a receptor-coupled transduction cascade signals temperature changes to the circadian clock. We initiated the further genetic dissection of temperature-entrainment and isolated the novel Drosophila mutation nocte, which is defective in molecular and behavioral entrainment by temperature cycles but synchronizes normally to light:dark cycles. CONCLUSIONS 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.

Loss of circadian clock function decreases reproductive fitness in males of Drosophila melanogaster.

Circadian coordination of life functions is believed to contribute to an organisms fitness; however, such contributions have not been convincingly demonstrated in any animal. The most significant measure of fitness is the reproductive output of the individual and species. Here we examined the consequences of loss of clock function on reproductive fitness in Drosophila melanogaster with mutated period (per0), timeless (tim0), cycle (cyc0), and Clock (ClkJrk) genes. Single mating among couples with clock-deficient phenotypes resulted in ≈40% fewer progeny compared with wild-type flies, because of a decreased number of eggs laid and a greater rate of unfertilized eggs. Male contribution to this phenotype was demonstrated by a decrease in reproductive capacity among per0 and tim0 males mated with wild-type females. The important role of clock genes for reproductive fitness was confirmed by reversal of the low-fertility phenotype in flies with rescued per or tim function. Males lacking a functional clock showed a significant decline in the quantity of sperm released from the testes to seminal vesicles, and these tissues displayed rhythmic and autonomous expression of clock genes. By combining molecular and physiological approaches, we identified a circadian clock in the reproductive system and defined its role in the sperm release that promotes reproductive fitness in D. melanogaster.

Drosophila cryptochromes: A unique circadian-rhythmphotoreceptor

Cryptochrome proteins are critical for circadian rhythms, but their function(s) is uncertain. Here we show that a mutation in a cryptochrome (dCRY) from the fruitfly Drosophila blocks an essential photoresponse of circadian rhythms, namely arrhythmicity under constant light conditions. We conclude that dCRY acts as a key photoreceptor for circadian rhythms and that there is probably no other comparable photoreceptor in this species.

Novel Features of Drosophila period Transcription Revealed by Real-Time Luciferase Reporting

The rapid turnover of luciferase and the sensitive, non-invasive nature of its assay make this reporter gene uniquely situated for temporal gene expression studies. To determine the in vivo regulatory pattern of the Drosophila clock gene period (per), we generated transgenic strains carrying a luciferase cDNA fused to the promoter region of the per gene. This has allowed us to monitor circadian rhythms of bioluminescence from pacemaker cells within the head for several days in individual living adults. These high time-resolution experiments permitted neuronal per transcription and opens the door to vastly simplified experiments in general chronobiology and studies of temporally regulated transcription in a wide range of experimental systems.

Circadian Photoreception in Drosophila: Functions of Cryptochrome in Peripheral and Central Clocks

In Drosophila melanogaster, disruption of night by even short light exposures results in degradation of the clock protein TIMELESS (TIM), leading to shifts in the fly molecular and behavioral rhythms. Several lines of evidence indicate that light entrainment of the brain clock involves the blue-light photoreceptor cryptochrome (CRY). In cryptochrome-depleted Drosophila (cry b ), the entrainment of the brain clock by short light pulses is impaired but the clock is still entrainable by light-dark cycles, probably due to light input from the visual system. Whether cryptochrome and visual transduction pathways play a role in entrainment of noninnervated, directly photosensitive peripheral clocks is not known and the subject of this study. The authors monitored levels of the clock protein TIM in the lateral neurons (LNs) of larval brains and in the renal Malpighian tubules (MTs) of flies mutant for the cryptochromegene (cry b ) and in mutants that lack signaling from the visual photopigments (norpA P 41). In cry b flies, light applied during the dark period failed to induce degradation of TIM both in MTs and in LNs, yet attenuated cycling of TIM was observed in both tissues in LD. This cycling was abolished in LNs, but persisted in MTs, of norpA P 41;cry b double mutants. Furthermore, the activity of the tim gene in the MTs of cry b flies, reported by luciferase, seemed stimulated by lights-on and suppressed by lights-off, suggesting that the absence of functional cryptochrome uncovered an additional light-sensitive pathway synchronizing the expression of TIM in this tissue. In constant darkness, cycling of TIM was abolished in MTs; however, it persisted in LNs of cry b flies. The authors conclude that cryptochrome is involved in TIM-mediated entrainment of both central LN and peripheral MT clocks. Cryptochrome is also an indispensable component of the endogenous clock mechanism in the examined peripheral tissue, but not in the brain. Thus, although neural and epithelial cells share the core clock mechanism, some clock components and light-entrainment pathways appear to have tissue-specific roles.

Cryptochrome Is Present in the Compound Eyes and a Subset of Drosophila's Clock Neurons

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 Drosophilas 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. J. Comp. Neurol. 508:952–966, 2008.