InteractiveFly: GeneBrief

cryptochrome: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - cryptochrome

Synonyms - Blue-light receptor

Cytological map position - 92A1--92A3

Function - circadian photoreceptor, light-responsive flavoprotein, potential transcription factor

Keywords - photoperiod response, enzyme, ventral midline

Symbol - cry

FlyBase ID:FBgn0025680

Genetic map position - 3-

Classification - cryptochrome/photolyase

Cellular location - unknown, presumably nuclear



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Yoshii, T., Hermann-Luibl, C., Kistenpfennig, C., Schmid, B., Tomioka, K. and Helfrich-Forster, C. (2015). Cryptochrome-dependent and -independent circadian entrainment circuits in Drosophila. J Neurosci 35: 6131-6141. PubMed ID: 25878285
Summary:
Entrainment to environmental light/dark (LD) cycles is a central function of circadian clocks. In Drosophila, entrainment is achieved by Cryptochrome (Cry) and input from the visual system. During activation by brief light pulses, Cry triggers the degradation of Timeless and subsequent shift in circadian phase. This is less important for LD entrainment, leading to questions regarding light input circuits and mechanisms from the visual system. Recent studies show that different subsets of brain pacemaker clock neurons, the morning (M) and evening (E) oscillators, have distinct functions in light entrainment. However, the role of Cry in M and E oscillators for entrainment to LD cycles is unknown. This study addresses this question by selectively expressing Cry in different subsets of clock neurons in a cry-null (cry0) mutant background. It was possible to rescue the light entrainment deficits of cry0 mutants by expressing Cry in E oscillators but not in any other clock neurons. Par domain protein 1 molecular oscillations in the E, but not M, cells of cry0 mutants still responded to the LD phase delay. This residual light response was stemming from the visual system because it disappeared when all external photoreceptors were ablated genetically. It is concluded that the E oscillators are the targets of light input via Cry and the visual system and are required for normal light entrainment.

Alex, A., et al. (2015). A circadian clock gene, cry, affects heart morphogenesis and function in Drosophila as revealed by optical coherence microscopy. PLoS One 10: e0137236. PubMed ID: 26348211
Summary:
Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues. The role that circadian clock genes play in heart development and function are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. This study performed analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, dCry was silenced by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, Wingless. Collectively, these studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function.

Qin, S., Yin, H., Yang, C., Dou, Y., Liu, Z., Zhang, P., Yu, H., Huang, Y., Feng, J., Hao, J., Hao, J., Deng, L., Yan, X., Dong, X., Zhao, Z., Jiang, T., Wang, H. W., Luo, S. J. and Xie, C. (2015). A magnetic protein biocompass. Nat Mater [Epub ahead of print]. PubMed ID: 26569474
Summary:
The notion that animals can detect the Earth's magnetic field was once ridiculed, but is now well established. Yet the biological nature of such magnetosensing phenomenon remains unknown. This study reports a putative magnetic receptor (Drosophila CG8198, here named MagR) and a multimeric magnetosensing rod-like protein complex, identified by theoretical postulation and genome-wide screening, and validated with cellular, biochemical, structural and biophysical methods. The magnetosensing complex consists of the identified putative magnetoreceptor and known magnetoreception-related photoreceptor cryptochromes, has the attributes of both Cry- and iron-based systems, and exhibits spontaneous alignment in magnetic fields, including that of the Earth. Such a protein complex may form the basis of magnetoreception in animals, and may lead to applications across multiple fields.

Bae, J. E., Bang, S., Min, S., Lee, S. H., Kwon, S. H., Lee, Y., Lee, Y. H., Chung, J. and Chae, K. S. (2016). Positive geotactic behaviors induced by geomagnetic field in Drosophila. Mol Brain 9: 55. PubMed ID: 27192976
Summary:
Appropriate vertical movement is critical for the survival of flying animals. Although negative geotaxis (moving away from Earth) driven by gravity has been extensively studied, much less is understood concerning a static regulatory mechanism for inducing positive geotaxis (moving toward Earth). Using Drosophila melanogaster as a model organism, this study showed that geomagnetic field (GMF) induces positive geotaxis and antagonizes negative gravitaxis. Remarkably, GMF acts as a sensory cue for an appetite-driven associative learning behavior through the GMF-induced positive geotaxis. This GMF-induced positive geotaxis requires the three geotaxis genes, such as cry, the cation channel pyx and pdf, and the corresponding neurons residing in Johnston's organ of the fly's antennae. These findings provide a novel concept with the neurogenetic basis on the regulation of vertical movement by GMF in the flying animals.

Ganguly, A., Manahan, C. C., Top, D., Yee, E. F., Lin, C., Young, M. W., Thiel, W. and Crane, B. R. (2016). Changes in active site histidine hydrogen bonding trigger cryptochrome activation. Proc Natl Acad Sci U S A 113: 10073-10078. PubMed ID: 27551082
Summary:
Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.
Schlichting, M., Menegazzi, P. and Helfrich-Forster, C. (2015). Normal vision can compensate for the loss of the circadian clock. Proc Biol Sci 282 [Epub ahead of print]. PubMed ID: 26378222
Summary:
Circadian clocks are thought to be essential for timing the daily activity of animals, and consequently increase fitness. This view was recently challenged for clock-less fruit flies and mice that exhibited astonishingly normal activity rhythms under outdoor conditions. Compensatory mechanisms appear to enable even clock mutants to live a normal life in nature. This study showed that gradual daily increases/decreases of light in the laboratory suffice to provoke normally timed sharp morning (M) and evening (E) activity peaks in clock-less flies. It was also shown that the compound eyes, but not Cryptochrome (Cry), mediate the precise timing of M and E peaks under natural-like conditions, as Cry-less flies do and eyeless flies do not show these sharp peaks independently of a functional clock. Nevertheless, the circadian clock appears critical for anticipating dusk, as well as for inhibiting sharp activity peaks during midnight. Clock-less flies only increase E activity after dusk and not before the beginning of dusk, and respond strongly to twilight exposure in the middle of the night. Furthermore, the circadian clock responds to natural-like light cycles, by slightly broadening Timeless (Tim) abundance in the clock neurons, and this effect is mediated by Cry.
Baik, L.S., Fogle, K.J., Roberts, L., Galschiodt, A.M., Chevez, J.A., Recinos, Y., Nguy, V. and Holmes, T.C. (2017). Cryptochrome mediates behavioral choice in response to UV light. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 28062690
Summary:
Drosophila melanogaster Cryptochrome (CRY) mediates behavioral and electrophysiological responses to blue light coded by circadian and arousal neurons. However, spectroscopic and biochemical assays of heterologously expressed CRY suggest that CRY may mediate functional responses to UV-A (ultraviolet A) light as well. To determine the relative contributions of distinct phototransduction systems, this study tested mutants lacking CRY and mutants with disrupted opsin-based phototransduction for behavioral and electrophysiological responses to UV light. CRY and opsin-based external photoreceptor systems cooperate for UV light-evoked acute responses. CRY mediates behavioral avoidance responses related to executive choice, consistent with its expression in central brain neurons.

Arthaut, L. D., et al. (2017). Blue-light induced accumulation of reactive oxygen species is a consequence of the Drosophila cryptochrome photocycle. PLoS One 12(3): e0171836. PubMed ID: 28296892
Summary:
Cryptochromes are evolutionarily conserved blue-light absorbing flavoproteins which participate in many important cellular processes including in entrainment of the circadian clock. Drosophila cryptochrome (DmCry) absorbs light through a flavin (FAD) cofactor that undergoes photoreduction to the anionic radical (FAD*-) redox state both in vitro and in vivo. However, recent efforts to link this photoconversion to the initiation of a biological response have remained controversial. By kinetic modeling of the DmCry photocycle this study shows that the fluence dependence, quantum yield, and half-life of flavin redox state interconversion are consistent with the anionic radical (FAD*-) as the signaling state in vivo. Fluorescence detection techniques showed that illumination of purified DmCry results in enzymatic conversion of molecular oxygen (O2) to reactive oxygen species (ROS). These observations were extended in living cells to demonstrate transient formation of superoxide (O2*-), and accumulation of hydrogen peroxide (H2O2) in the nucleus of insect cell cultures upon DmCry illumination. These results define the kinetic parameters of the Drosophila cryptochrome photocycle and support light-driven electron transfer to the flavin in DmCry signaling. They furthermore raise the intriguing possibility that light-dependent formation of ROS as a byproduct of the cryptochrome photocycle may contribute to its signaling role.
Menegazzi, P., Dalla Benetta, E., Beauchamp, M., Schlichting, M., Steffan-Dewenter, I. and Helfrich-Förster, C. (2017). Adaptation of circadian neuronal network to photoperiod in high-latitude European Drosophilids. Curr Biol 27: 833-839. PubMed ID: 28262491
Summary:
The genus Drosophila contains over 2,000 species that, stemming from a common ancestor in the Old World Tropics, populate today very different environments. This study found significant differences in the activity pattern of Drosophila species belonging to the holarctic virilis group, i.e., D. ezoana and D. littoralis, collected in Northern Europe, compared to that of the cosmopolitan D. melanogaster, collected close to the equator. These behavioral differences might have been of adaptive significance for colonizing high-latitude habitats and hence adjust to long photoperiods. Most interestingly, the flies' locomotor activity correlates with the neurochemistry of their circadian clock network, which differs between low and high latitude for the expression pattern of the blue light photopigment cryptochrome (CRY) and the neuropeptide Pigment-dispersing factor (PDF). In D. melanogaster, CRY and PDF are known to modulate the timing of activity and to maintain robust rhythmicity under constant conditions. The rhythmic behavior of the high-latitude virilis group species could be partially stimulated by mimicking their CRY/PDF expression patterns in a laboratory strain of D. melanogaster. Data suggest that these alterations in the CRY/PDF clock neurochemistry might have allowed the virilis group species to colonize high-latitude environments.

Damulewicz, M., Mazzotta, G. M., Sartori, E., Rosato, E., Costa, R. and Pyza, E. M. (2017). Cryptochrome is a regulator of synaptic plasticity in the visual system of Drosophila melanogaster. Front Mol Neurosci 10: 165. PubMed ID: 28611590
Summary:
Drosophila Cryptochrome (Cry) is a blue light sensitive protein with a key role in circadian photoreception. A main feature of Cry is that light promotes an interaction with the circadian protein Timeless (Tim) resulting in their ubiquitination and degradation, a mechanism that contributes to the synchronization of the circadian clock to the environment. Moreover, Cry participates in non-circadian functions such as magnetoreception, modulation of neuronal firing, phototransduction and regulation of synaptic plasticity. This study used co-immunoprecipitation, yeast 2 hybrid (Y2H) and in situ proximity ligation assay (PLA) to show that Cry can physically associate with the presynaptic protein Bruchpilot (Brp) and that Cry-Brp complexes are located mainly in the visual system. Additionally, evidence is presented that light-activated Cry may decrease Brp levels in photoreceptor termini in the distal lamina, probably targeting Brp for degradation.
Agrawal, P., Houl, J. H., Gunawardhana, K. L., Liu, T., Zhou, J., Zoran, M. J. and Hardin, P. E. (2017). Drosophila CRY entrains clocks in body tissues to light and maintains passive membrane properties in a non-clock body tissue independent of light. Curr Biol [Epub ahead of print]. PubMed ID: 28781048
Summary:
Circadian clocks regulate daily rhythms in physiology, metabolism, and behavior via cell-autonomous transcriptional feedback loops. In Drosophila, the blue-light photoreceptor Cryptrochrome (CRY) synchronizes these feedback loops to light:dark cycles by binding to and degrading Timeless (TIM) protein. CRY also acts independently of TIM in Drosophila to alter potassium channel conductance in arousal neurons after light exposure, and in many animals CRY acts independently of light to repress rhythmic transcription. CRY expression has been characterized in the Drosophila brain and eyes, but not in peripheral clock and non-clock tissues in the body. To investigate CRY expression and function in body tissues, a GFP-tagged-cry transgene was generated that rescues light-induced behavioral phase resetting in cry03 mutant flies and sensitively reports GFP-CRY expression. In bodies, CRY is detected in clock-containing tissues including Malpighian tubules, where it mediates both light-dependent TIM degradation and clock function. In larval salivary glands, which lack clock function but are amenable to electrophysiological recording, CRY prevents membrane input resistance from falling to low levels in a light-independent manner. The ability of CRY to maintain high input resistance in these non-excitable cells also requires the K+ channel subunits Hyperkinetic, Shaker, and ether-a-go-go. These findings for the first time define CRY expression in Drosophila peripheral tissues and reveal that CRY acts together with K+ channels to maintain passive membrane properties in a non-clock-containing peripheral tissue independent of light.
Damulewicz, M., Loboda, A., Jozkowicz, A., Dulak, J. and Pyza, E. (2017). Haeme oxygenase protects against UV light DNA damages in the retina in clock-dependent manner. Sci Rep 7(1): 5197. PubMed ID: 28701782
Summary:
This study showed that in the retina of Drosophila, the expression of the Ho gene, encoding Heme oxygenase (Ho), is regulated by light but only at the beginning of the day. This timing must be set by the circadian clock, as light pulses applied at other time points during the day do not increase the ho mRNA level. Moreover, light-induced activation of HO does not depend on the canonical phototransduction pathway but instead involves Cryptochrome and is enhanced by ultraviolet (UV) light. Interestingly, the level of DNA damage in the retina after UV exposure was inversely related to the circadian oscillation of the Ho mRNA level during the night, being the highest when the Ho level is low and reverses during the day. Accordingly, induction of Ho by the iron containing metalloporphyrin hemin was associated with low DNA damage, while inhibition of Ho activity by SnPPIX aggravated the damage. These data suggest that Ho acts in the retina to decrease oxidative DNA damage in photoreceptors caused by UV-rich light in the morning.
Agrawal, P., Houl, J. H., Gunawardhana, K. L., Liu, T., Zhou, J., Zoran, M. J. and Hardin, P. E. (2017). Drosophila CRY entrains clocks in body tissues to light and maintains passive membrane properties in a non-clock body tissue independent of light. Curr Biol [Epub ahead of print]. PubMed ID: 28781048
Summary:
Circadian clocks regulate daily rhythms in physiology, metabolism, and behavior via cell-autonomous transcriptional feedback loops. In Drosophila, the blue-light photoreceptor Cryptochrome (Cry) synchronizes these feedback loops to light:dark cycles by binding to and degrading Timeless (Tim) protein. CRY also acts independently of TIM in Drosophila to alter potassium channel conductance in arousal neurons after light exposure, and in many animals CRY acts independently of light to repress rhythmic transcription. CRY expression has been characterized in the Drosophila brain and eyes, but not in peripheral clock and non-clock tissues in the body. To investigate CRY expression and function in body tissues, a GFP-tagged-cry transgene was generated that rescues light-induced behavioral phase resetting in cry03 mutant flies and sensitively reports GFP-CRY expression. In bodies, CRY is detected in clock-containing tissues including Malpighian tubules, where it mediates both light-dependent TIM degradation and clock function. In larval salivary glands, which lack clock function but are amenable to electrophysiological recording, CRY prevents membrane input resistance from falling to low levels in a light-independent manner. The ability of CRY to maintain high input resistance in these non-excitable cells also requires the K+ channel subunits Hyperkinetic, Shaker, and Ether-a-go-go. These findings for the first time define CRY expression in Drosophila peripheral tissues and reveal that CRY acts together with K+ channels to maintain passive membrane properties in a non-clock-containing peripheral tissue independent of light.
BIOLOGICAL OVERVIEW

In Drosophila, genetic ablation of eyes or mutations in the well-characterized visual phototransduction pathway do not block entrainment of circadian clocks to light. In other words, blind flies still exhibit a photoperiod response. For example, Drosophila mutants norpA (norpA codes for phospholipase C involved in visual phototransduction) and ninaE (ninaE codes for the Rh1 rhodopsin visual pigment in rhabdomeres R1-R6) still have robust and light-sensitive rhythms (Dushay, 1989; Zerr, 1990; Wheeler, 1993; Suri, 1998 and Yang, 1998), indicating that there is at least one additional relevant photoreceptor and signal transduction pathway independent of the now classical photoperiod transcription factors, Period, Timeless, Clock, and Cycle. The existence of such a pathway finds support with the cloning of the cryptochrome (cry) gene, coding for a cryptochrome/photolyase, a member of a flavin based light-intercepting protein family whose members have been previously implicated as sensors of DNA damage. Drosophila Cry may be implicated in DNA repair activity. The relevant residues are well conserved within the family, but some members with comparable conservation (mammalian cryptochromes, for example) fail to manifest repair activity. Members of this family bind DNA and have characteristics of transcription factors. All characterized family members are directly photosensitive and include plant blue light photoreceptors. cry transcription is under circadian regulation, influenced by the Drosophila clock genes period, timeless, Clock, and cycle. Cry protein levels are dramatically affected by light exposure. Importantly, circadian photosensitivity is increased in a cry-overexpressing strain. These physiological and genetic data therefore link a specific photoreceptor molecule to circadian rhythmicity. It has been proposed that Cry is a major Drosophila photoreceptor dedicated to the resetting of circadian rhythms (Emery, 1998 and Stanewsky, 1998).

The characteristics of this additional pathway can be addressed using action spectra. Action spectra have previously been used to measure phase response curves (PRCs) for Drosophila pseudoobscura eclosion; in these earlier studies, the PRCs peaked at 400-500 nm (Frank, 1969 and Klemm, 1976). Only recently have similar Drosophila melanogaster action spectra been derived for adult locomotor activity rhythms and for TIM degradation (Suri, 1998). Both curves are significantly different from that of Drosophila vision, suggesting a novel blue light circadian photoreceptor that affects behavioral rhythms as well as the response of peripheral clocks, even in the eye. In mammals, the retina is very important for circadian light perception and makes synaptic connections with the suprachiasmatic nucleus via the retino-hypothalamic tract. But the relevant photoreceptor(s) is uncertain, since the well-studied mammalian visual photopigments may be insufficient to account for ocular circadian photoreception (Foster, 1998). Therefore, as in Drosophila, the mammalian eye may contain another unidentified photoreceptor specialized for circadian light perception (Miyamoto, 1998 and Soni, 1998).

Given the action spectrum, it has been suggested that the unknown Drosophila photoreceptor might be a member of the photolyase/cryptochrome family, a well-characterized group of blue light-responsive flavoproteins. Photolyases/cryptochromes have five well-characterized subgroups (Kanai, 1997). Three groups of cyclobutane pyrimidine dimers (CPD) photolyases are responsible for the repair of thymidine dimers caused by UV irradiation. Another group contains the 6-4 photolyases, as found in Drosophila (Todo, 1996), responsible for the repair of UV-generated 6-4 thymidine dimers and 6-4 photolyase homologs. A fifth group contains plant blue light photoreceptors (Ahmad, 1993 and Lin, 1998). Evidence from several systems suggests that a flavin-based system, in addition to or instead of retinal-containing proteins, contributes to circadian photoreception. Caroteinoid-depleted Drosophila show normal photosensitivity (Zimmerman, 1971). In Neurospora, a genetic defect in the riboflavin synthesis pathway inhibits the photoentrainment pathway. In Arabidopsis, two cryptochromes have been shown to be involved in three different light-dependent processes: hypocotyl elongation inhibition, phototropism, and photoperiodism of flowering time (Ahmad, 1998a; Guo, 1998; Lin, 1998). Flowering time photoperiodism may be related to circadian rhythms (Guo, 1998). In mammals, a recent report describes the expression pattern of two novel cryptochromes, mCRY1 and mCRY2 (Miyamoto, 1998). The transcripts are expressed in retina and the suprachiasmatic nucleus, tissues highly relevant to circadian rhythms. Moreover, mcry1 transcript undergoes circadian cycling in the SCN. Although there is no evidence that the SCN is directly light sensitive, the authors propose that these cryptochromes are involved in mammalian circadian photoreception. Importantly, there are no physiological data that link these or any other specific cryptochromes to circadian light perception in any animal system (Emery, 1998).

A property shared by many clock gene transcripts is that their abundance is subject to circadian oscillation. To assay CRY mRNA, Northern and RNase protection analyses were performed on RNA isolated from heads after fly entrainment under standard light/dark conditions (LD). Both assays gave identical results: CRY mRNA manifests ~5-fold amplitude cycling, with a peak at ZT1-5 (1 to 5 hours after the lights go on) and a trough at ZT17 (five hours into the dark part of the light/dark cycle). A higher resolution RNase protection experiment reveals that the peak persists from ZT1 to ZT7, and the trough from ZT17 to ZT19. CRY mRNA cycling was also measured under conditions of constant darkness. Cycling persists under these conditions but with a lower, approximately 2-fold amplitude. Lower amplitude cycling in constant darkness has been previously described for other clock genes or for clock gene-derived reporter genes. Because there is good evidence for peripheral clocks in multiple tissues throughout Drosophila, RNA extracted from fly bodies was also examined. CRY body mRNA cycling has a similar phase to head mRNA cycling but only a 2.5-fold amplitude. There is precedence for lower amplitude clock mRNA cycling in bodies as compared to heads (Emery, 1998 and references).

CRY mRNA cycling suggests that the protein abundance also cycles during the day, as shown previously for Per and Tim, and very recently for Clk. To address this question, a rat antibody directed against the N terminus of Cry was used in Western blotting experiments. The antibody shows specific immunoreactivity to in vitro translated Cry. The very strong signal obtained with extracts from heat-shocked flies containing the cry gene under hsp70 promoter control shows that the antibody also specifically recognizes Cry in head extract. The protein migrates with an apparent molecular weight of approximately 60 kDa, as predicted from the cDNA sequence. Two bands were visible on some gels, whereas a single fuzzy band was detected on others. The protein abundance was measured in light/dark entrained wild-type (Canton-S) flies, as well as in y w;timgal4 flies (flies with white, pigmentless eyes). The latter show a very robust 8-fold amplitude cycle, which is strikingly different from the CRY mRNA cycle. Protein is low during the day when the mRNA is high, clearly increases before the mRNA rise, and peaks at ZT23 (an hour before the beginning of the lights on part of the cycle). Two hours later, at ZT1, when the CRY mRNA peaks, Cry protein is already strongly reduced (50% of peak value). This indicates that a translational or posttranslational mechanism makes a strong contribution to Cry regulation. In wild-type Canton-S flies, the cycle was less robust (3- to 5-fold), and the trough is reached between ZT11 and ZT13. Between ZT1 and ZT9, protein levels are approximately 40%-50% of the peak. Lower amplitude Cry cycling in CS versus y w flies is probably due to light shielding by eye pigment, as described for the Tim light response (Suri, 1998 and Emery, 1998).

To determine whether the Cry protein cycling is light driven (for example, if the Cry protein itself is denatured by light), protein levels were measured under constant dark (DD) conditions. Surprisingly, and in sharp contrast with Per, Tim, and Clk, the Cry DD pattern is completely different from that in LD and increases continuously in constant darkness throughout the subjective day and night, beginning at the levels reached at ZT23 of the previous day. In DD conditions as well as in LD conditions, the protein and mRNA profiles are completely different. These results strongly suggest that Cry cycling is in great part under light control. To further address the role of light, Cry protein levels were examined in per0, tim0, Clk, and cyc0 arrhythmic backgrounds. As expected, relative protein levels correlate with the relative RNA levels, higher in Clk and cyc0 than in per0 and tim0. But there was also robust Cry cycling in all these clock mutants, in contrast to what was observed for CRY mRNA cycling under the same LD conditions. Taken together, the results show that Cry expression is light regulated at the translational or posttranslational level as well as clock regulated at the transcriptional level (Emery, 1998).

It is presumed that photon capture by Cry itself increases proteolysis, and the data suggest that Cry is very stable at night and unstable during the day. Perhaps the CRY mRNA oscillation is important for the proper increase and decrease in Cry levels that take place after lights off and lights on, respectively. These might be more pronounced or more important during the gradual changes in illumination that occur during natural light-dark cycles. Interestingly, Arabidopsis CRY2 is also light sensitive and accumulates at low light intensities (Ahmad, 1998b and Lin, 1998).

Light-mediated changes in Cry levels may be important for signal transduction downstream of Cry, or they may occur subsequent to signal transduction. Signaling may take place by electron transfer, protein-protein contact, or by changes in phosphorylation state. These possibilities are just beginning to be explored in the case of plant and animal cryptochromes (Huala, 1997; Zhao, 1997; Ahmad, 1998a). But the ultimate targets in the case of Drosophila Cry are likely to be clock molecules. Indeed, Tim levels are also light sensitive, and there is evidence that this molecular light response is relevant to the behavioral light response (Suri, 1998 and Yang, 1998). Since Tim is unresponsive in the cryb mutant [a recessive third chromosomal mutation that abolishes bioluminescence rhythms, identified by Stanewsky (1998)] and Cry light regulation is independent of other clock molecules, including Tim, this establishes a clear epistatic relationship between Cry and Tim. Yet CRY mRNA cycling is clock regulated, indicating feedback between the central pacemaker and this clock input component. Conservation of the putative DNA-binding residues in Drosophila suggests another, somewhat audacious possibility: Cry might be a DNA-binding protein and function as a transcriptional regulator of light-induced gene expression. Subcellular localization and mutagenesis studies should aid in determining whether Cry-DNA binding is relevant to the light response in Drosophila. In mammals, mper1 transcription is rapidly induced by light in a rhythm-relevant manner and there are other data that link immediate early gene expression to phase-shifting light pulses. mCRY1 and 2 have not been shown to be functionally relevant to rhythms or to rhythm-relevant immediate early gene expression, and no light-regulated genes are known in Drosophila. But these differences are probably temporary, indicating that Cry might well connect to transcription in both systems. As Cry autoregulates expression (Stanewsky, 1998), it is even possible that it participates directly, or once participated directly, in its own transcriptional regulation. Photoreception and transcriptional feedback loops are ubiquitous features of circadian pacemakers. Therefore, circadian rhythms may have begun long ago when a DNA repair protein acquired the ability to autoregulate expression in a light- and ultimately time-dependent manner (Emery, 1998 and references).

cryb is an apparent null mutation in a gene encoding Drosophila's version of the blue light receptor Cryptochrome. The mutation maps to a position approximately in the middle of the right arm of chromosome 3. Flies heterozygous for the mutation exhibit poor bioluminescence rhythms. Although a higher proportion of mutation-over-deletion males is rhythmic, as compared to homozygous mutant flies, formal analysis of these rhythms indicates that they are very weak and exhibit anomalous phases; mutation-over-deletion females are thoroughly arrhythmic (Stanewsky, 1998).

Given the drastic effects of this mutation on cyclic expression of Per and Tim proteins (see cryptochrome Targets of Activity), it was expected that the locomotor behavior of cryb would also be abnormal. Surprisingly, cryb flies are rhythmic in both LD and DD conditions, given ~24 hr periods. In further LD experiments, flies were first exposed to 5 days of 12 hr:12 hr LD (white light, intensity 640 lux), followed by a second 5 day LD regime in which the lights came on 4 hr later and were changed to dim blue. cryb flies entrain to the initial LD cycles, since all the mutant individuals exhibit characteristic bimodal activity peaks. These mutant flies also have no trouble reentraining to the second light regimes, even though a much lower light intensity was used. The same behavior is observed for cry+ and cryb flies at all light intensities (Stanewsky, 1998).

Externally blind flies behaviorally entrain to LD cycles but are less sensitive to the synchronizing effects of light in terms of their behavioral rhythmicity. Hence, the light responsiveness of norpAP41;cryb double mutants was tested. norpA codes for a phospholipase C (PLC) activity involved in the phototransduction cascade that takes place within the eye. The norpA loss-of-function mutation causes the compound eyes and ocelli to be completely unresponsive to light (Pearn, 1996). The doubly mutant flies exhibit entrainment problems in the initial LD regime. Subsequently, only about 50% of the flies that entrained to the initial LD cycle are able to synchronize to a new light regime that applies 16-lux blue light; and at still lower light intensities, nearly all of the double mutants fail to entrain to the new LD cycles. Since norpA and cryb mutations by themselves cause minimal or no entrainment problems at these low light intensities, the synergistic effect observed in the double mutant suggests that there are two light entrainment pathways operating in Drosophila's rhythm system. A fast clock period mutation also uncovers entrainment defects in cryb mutants (Stanewsky, 1998).

The behavioral effects of cryb indicate that the normal function of the gene is involved in light-mediated entrainment and the ability to reset the clock pacemaker that underlies Drosophila's daily rhythms of locomotion. Yet the protein encoded by cry is not the only photoreceptor involved in this process: rhodopsin molecules function upstream of the NorpA PLC protein in the canonical phototransduction cascade that is a part of the input pathway to the fly's clock. PLC could be functioning within the anatomical elements of such pathways located in the CNS as well as within the external eyes, because norpA's PLC product is found within the brain as well as in the eyes. This PLC could even act downstream of cry in extraocular locations, but if this were the case, a norpA mutation would be sufficient to block entrainment in medium-dim light, and this is not so. Therefore, the combined effects of norpA and cryb mutations on entrainment are likely to be the result of these two functions acting separately in the eye and in the CNS, respectively. This means that there are anatomically independent light input pathways to the pacemaker that control behavior. One of these might involve brain neurons that feed light into the clock. Alternatively, the extraocular pathway could function intracellularly within clock neurons themselves, by analogy to pinealocytes in lower vertebrates being both circadian pacemaker cells and photoreceptors. But the fact that anatomical eyelessness causes the same decrement in circadian light sensitivity as do norpA mutations suggests that the latter's effects are acting only through external photoreceptors. In this scenario, norpA would participate merely as part of an eye-to-brain throughput pathway, via the compound eyes and optic ganglia, eventually reaching the CNS pacemaker. The fact that Cry is not the only important input factor in the fly's circadian system fits with results from other systems, ranging from microbes to mammals. Multiple input pathways operate with regard to both anatomical structures and physiological processes. The adaptive value of such complexity is that it permits entraining organisms to sample light of several different wavelengths. Indeed, the quality of light reaching the earth's surface changes dramatically from dawn and dusk, in particular being enriched in the blue wavelengths during the dimmest portions of these daily transition times (Stanewsky, 1998 and references)

Cryptochrome mediates light-dependent magnetosensitivity in Drosophila

Although many animals use the Earth's magnetic field for orientation and navigation, the precise biophysical mechanisms underlying magnetic sensing have been elusive. One theoretical model proposes that geomagnetic fields are perceived by chemical reactions involving specialized photoreceptors. However, the specific photoreceptor involved in such magnetoreception has not been demonstrated conclusively in any animal. This study shows that the ultraviolet-A/blue-light photoreceptor cryptochrome (Cry) is necessary for light-dependent magnetosensitive responses in Drosophila. In a binary-choice behavioural assay for magnetosensitivity, wild-type flies show significant naive and trained responses to a magnetic field under full-spectrum light (~300-700 nm) but do not respond to the field when wavelengths in the Cry-sensitive, ultraviolet-A/blue-light part of the spectrum (<420 nm) are blocked. Notably, Cry-deficient cry0 and cryb flies do not show either naive or trained responses to a magnetic field under full-spectrum light. Moreover, Cry-dependent magnetosensitivity does not require a functioning circadian clock. This work provides the first genetic evidence for a Cry-based magnetosensitive system in any animal (Gegear, 2008).

The ability of an animal to detect geomagnetic fields has substantial biological relevance as it is used by many invertebrate and vertebrate species for orientation and navigation purposes, including homing, building activity and long-distance migration. Three general modes of magnetoreception have been proposed. One mode is electromagnetic induction by the Earth's magnetic field, which may occur in electrosensitive marine fish, although there is little evidence to support such sensing. The two other modes, for which experimental evidence does exist, are a magnetite-based process and chemical-based reactions that are modulated by magnetic fields. One chemical model of magnetoreception proposes that magnetic information is transmitted to the nervous system through the light-induced product of magnetically sensitive radical-pair reactions in specialized photoreceptors (Gegear, 2008).

Cry proteins are flavoproteins that have been postulated to generate magnetosensitive radical pairs that could provide a photoinduced electron transfer reaction for the detection of magnetic fields. Cry proteins are best known for their roles in the regulation of circadian clocks and can be categorized into two groups on the basis of current phylogenetic and functional relationships. Drosophila-like Cry proteins are sensitive to light in the ultraviolet-A/blue range and function primarily as photoreceptors that synchronize (entrain) circadian clocks. Vertebrate-like Cry proteins, which have also been found in every non-drosophilid insect so far examined, do not seem to be directly light-sensitive. Instead, vertebrate-like Cry proteins are potent repressors of the Clock and Bmal1 (known as Cycle in insects) transcription factors which, as heterodimers, drive the intracellular transcriptional feedback loop of the circadian clock mechanism in all animals studied (Gegear, 2008).

Although there is good behavioural evidence for the involvement of short-wavelength photoreceptors in the detection of a geomagnetic field, an essential link between Cry and magnetoreception has not been established in any animal. Fruit flies are ideally suited to investigate a role for Cry as a magnetoreceptor, because they only have the light-sensitive Cry in which the action spectrum peaks in the ultraviolet-A range (350-400 nm) with a plateau in the near blue range (430-450 nm). Notably, flies that lack Cry (cry0) or harbour the chemically induced missense cryb mutation can be used to evaluate the role of Cry in magnetosensitive responses (Gegear, 2008).

These studies were initiated by developing a behavioural assay for magnetosensitivity in Drosophila. In this illuminated apparatus, flies experience a magnetic field generated by an electric coil system and display their magnetosensitivity in a binary-choice T-maze. The two-coil system is ideal for behavioural studies of magnetosensitivity, because it produces a magnetic field on one side of the T-maze, while producing no field on the opposite side. This design eliminates non-magnetic differences such as heat generated by the electric coils between sides during test sessions. Flies were tested either for their response to the magnetic field in the naive state (naive group) or after a training session pairing the field with sucrose reward (trained group) (Gegear, 2008).

Wild-type Canton-S, white-eyed w;Canton-S, Oregon-R and Berlin-K strains all developed a learned preference for a magnetic field. The trained groups in the two Canton-S lines showed the greatest response to the field and were the only ones to show a naive avoidance of the field. Thus, Drosophila consistently show magnetosensitivity that varies in magnitude in a strain-dependent manner. The similarity of behavioural responses between red-eyed, wild-type Canton-S flies and white-eyed w;Canton-S flies shows that eye colour does not substantially alter behavioural responses to the magnetic field (Gegear, 2008).

Because wild-type Canton-S flies showed the most robust trained and naive responses of the strains tested, they were used to determine whether the magnetic responses observed were light-dependent. Naive and trained Canton-S flies were trained under different long-wavelength pass filters that transmitted wavelengths of light at >500 nm, >420 nm or >400 nm. In contrast to flies assayed under full-spectrum light, flies did not show either naive or trained responses to the field when wavelengths <420 nm were blocked. Because the filter that blocked light <420 nm also caused a 13% decrease in total irradiance, whether the filter-induced lack of behavioural responses to the magnetic field was secondary to the decrement in irradiance was tested. When Canton-S flies were studied under full-spectrum light, with a total irradiance level lower than that imposed by the filter, the flies still showed significant naive and trained responses to the magnetic field. Thus, the filter-induced loss of behavioural responses to the magnetic field is due to the loss of short-wavelength light (Gegear, 2008).

Behavioural responses to the magnet were partially restored when 400-420 nm light was included, which is consistent with the action spectrum of Drosophila Cry tailing into the near blue, and, as expected, the trained response was weaker than that under full-spectrum light (full spectrum versus >400 nm). This wavelength-dependent effect of the magnetic field on behaviour suggests that Drosophila has a photoreceptor-based magnetosensitive system. Moreover, because the response to the magnetic field requires ultraviolet-A/blue light (<420 nm), these data are consistent with the hypothesis that Cry can function as a magnetoreceptor in Drosophila (Gegear, 2008).

Cry-deficient cry0 mutant flies were used to examine directly whether Cry is required for magnetosensitive behaviour. Two of the newly generated cry0 fly lines were tested, because, in cry0 flies, the entire cry coding sequence has been replaced with mini-white+ by homologous recombination, ensuring that, unlike in the more commonly used point-mutant cryb flies, there is no possibility of residual Cry activity. In addition, the three cry0 fly lines were backcrossed independently into a w1118 background. Thus, it was possible to use the appropriate w1118 control flies to test the contribution of the cry gene in magnetosensitive behaviour (Gegear, 2008).

Control w1118 flies showed a clear naive preference for, rather than avoidance of, the magnetic field. The difference in the direction of the naive response to the magnetic field between Canton-S flies and the w1118 line re-emphasizes the importance of controlling for genetic background in studies of magnetosensitivity in flies. Nonetheless, like Canton-S flies, the naive response of w1118 flies to the magnetic field was light dependent; the naive preference for the magnetic field was abolished in the absence of ultraviolet-A/blue light (Gegear, 2008).

Homozygous cry02 flies lacking Cry did not show a naive response to the magnet under full-spectrum light, in contrast to the significant naive responses manifested by both w1118 and heterozygous cry02/+ flies. Training control w1118 flies to prefer the magnetic field under full-spectrum light significantly enhanced their naive preference for the field. In contrast, homozygous cry01 flies did not show either a naive preference for the field (like cry02 flies) or an enhanced preference for the field after training. The loss of the response to the magnetic field in the Cry-deficient flies resembled the behaviour when w1118 flies were deprived of ultraviolet-A/blue light, which is consistent with Cry being the relevant light sensor. These data using two cry null strains strongly suggest that both naive and trained responses to the magnetic field in Drosophila require Cry function (Gegear, 2008).

The Cry-defective cryb mutant flies are also unable to respond to the magnetic field; the cryb mutation renders CryB essentially non-functional. Because the genetic background of cryb mutant flies is not well defined, behavioural responses were compared to the magnetic field between homozygous cryb flies and heterozygous cryb/Canton-S flies. Whereas homozygous cryb flies did not show either naive or trained responses to the magnetic field under full-spectrum light, heterozygous cryb/Canton-S flies showed significant naive and trained responses; the trained response in the heterozygotes was less than that of wild-type Canton-S flies and probably results from differences in genetic background (Gegear, 2008).

To rule out non-cry mutations as the reason for the lack of magnetic responses in cryb mutants, it was shown that the cryb mutation fails to complement the cry01 null mutation. Transheterozygous cryb/cry01 flies did not show significant naive or trained responses to the magnet, whereas heterozygous cry01/Canton-S and cryb/Canton-S flies did. Taken together, these data indicate that the cry locus is necessary for light-dependent magnetosensitivity in Drosophila. Furthermore, the lack of a trained response in both cry01 and cryb mutant flies is consistent with Cry being an essential component of the magnetosensitive sensory input pathway and perhaps the magnetoreceptor itself (Gegear, 2008).

Because light-activated Cry interacts with the critical circadian clock protein Timeless to reset the circadian clock mechanism, whether an intact circadian system is necessary for the Cry-dependent magnetosensitive responses was tested in wild-type Canton-S flies. Circadian arrhythmicity was induced by constant light, which disrupts circadian clock function in Cry-containing cells by causing the constant degradation of not only Cry but also Timeless and then Period. Subsequently behavioural responses were tested to the magnetic field after at least 5 days in constant light when the flies were shown to express arrhythmic locomotor behaviour, to have disrupted Period abundance rhythms, and to express constantly low levels of Cry. Notably, these arrhythmic flies continued to show significant naive and trained responses to the magnetic field. Thus, the continuous activation of Cry by light does not disrupt its ability to sense the magnet, and an intact circadian system is not required for the magnetoreception mechanism to operate (Gegear, 2008).

There are two other published reports of magnetosensitivity in adult Drosophila (Wehner, 1970; Phillips, 1993). One describes behavioural evidence that male wild-type Oregon-R flies show a light-dependent magnetic compass response in a radial maze whereas female flies did not respond to the magnet. Additionally, male flies responded in opposite directions when tested under either 365 nm or 500 nm light. In the studies, both male and female flies showed a magnetic response. Regardless of experimental differences, both the previous study (Phillips, 1993) and the current one demonstrate that fruitflies can respond to a magnetic field in a wavelength-dependent manner (Gegear, 2008).

The current results extend substantially the presence of a light-dependent magnetic sense in Drosophila by showing the necessity of Cry. It cannot bedistinguish unequivocally whether fly Cry functions as the actual magnetoreceptor or whether it is an essential component downstream of the receptor. Cry is necessary for both the naive and trained responses to the magnetic field, consistent with the notion that Cry is in the input pathway of magnetic sensing. In addition, the continued behavioural responses to the magnet in constant light, in which the known Cry signalling components are being constantly degraded and the circadian clock is rendered non-functional, is also consistent with an input function. The most compelling evidence supporting a magnetoreceptor role for Cry is that the Cry-dependent behavioural responses to the magnetic field require ultraviolet-A/blue light, which matches the action spectrum of (Gegear, 2008).

The behavioral assay for magnetosensitivity does not at present have a pure directional component, and therefore it is difficult to relate the findings directly to the use of geomagnetic fields for animal orientation and navigation. Nevertheless, it is probable that the identified response is the prototype for the involvement of Cry in chemical-based magnetic sensing. Thus, these findings open new avenues of investigation into the cellular and molecular basis of chemical-based magnetic sensing in animals. The powerful genetics of Drosophila will facilitate an understanding of the precise mechanism of action of Cry in magnetosensitivity, such as the actual involvement of magnetosensitive radical pairs produced by photoinduced electron transfer reactions. The data further show that the biological functions of Drosophila Cry extend beyond those in circadian clocks (Gegear, 2008).

CRYPTOCHROME is a blue-light sensor that regulates neuronal firing rate

Light-responsive neural activity in central brain neurons is generally conveyed through opsin-based signaling from external photoreceptors. Large lateral ventral arousal neurons (lLNvs) in Drosophila increase action potential firing within seconds in response to light in the absence of all opsin-based photoreceptors. Light-evoked changes in membrane resting potential occur in about 100 milliseconds. The light response is selective for blue wavelengths corresponding to the spectral sensitivity of Cryptochrome (Cry). cry-null lines are light-unresponsive, but restored Cry expression in the lLNv rescues responsiveness. Furthermore, expression of Cry in neurons that are normally unresponsive to light confers responsiveness. The Cry-mediated light response requires a flavin redox-based mechanism and depends on potassium channel conductance, but is independent of the classical circadian Cry-Timeless interaction (Fogle, 2011).

The Drosophila circadian clock circuit is composed of 140 to 150 neurons in the central brain and includes Pigment-dispersing factor (Pdf)-expressing lateral ventral neurons. The large lateral ventral neurons (lLNvs) are arousal neurons and increase spontaneous action potential firing in response to light, whereas the small lateral ventral neurons (sLNvs) are critical for circadian function. Light resets the circadian clock via two mechanisms: rhodopsin-based external photoreceptors [the compound eye, ocelli, and the Hofbauer-Buchner (HB) eyelet] and the blue-light photopigment Cryptochrome (Cry). Drosophila Cry is best known for its light-activated targeting of Tim for degradation, resetting the clock. External photoreceptors and Cry entrain the Drosophila circadian circuit at vanishingly low light levels. Cry also mediates magnetosensitivity in flies and butterflies (Fogle, 2011).

In addition to the circadian molecular clock, membrane excitability is a key component of normal maintenance of circadian rhythms. Electrophysiological characterization of the s- and lLNvs has shown that their membrane properties are circadian-regulated outputs as well. Spontaneous firing frequencies are higher during the early day, gradually drop until dusk, and then rise again through the course of the night. Additionally, the lLNv spontaneous firing frequency elevates 20% to 200% in response to moderately bright light. Given the plurality of light inputs to the lLNv, the lLNv electrophysiological light response was investigated and it was found that the response is due to Cry acting by a cell-autonomous, redox-based mechanism, independent of Cry-Tim interactions, which requires the conductance of membrane potassium channels. Furthermore, ectopic expression of Cry optogenetically confers electrophysiological light responsiveness to neurons that ordinarily do not respond to light (Fogle, 2011).

Both tonic and burst firing lLNvs recorded in the whole-cell current clamp configuration in an acutely dissected whole-brain preparation from flies expressing the pdfGAL4 driver and green fluorescent protein (GFP)-tagged nonconducting UAS-dORK, a Drosophila membrane-delimited potassium channel, (pdfGAL4-NC1-GFP) under dark conditions immediately increased their firing rate and their resting membrane potential in response to moderate-intensity white light or high-intensity blue light from a mercury light source, then rapidly returned to baseline firing rate upon return to darkness. The strength of the firing frequency lLNv light response, expressed as the firing frequency with the lights on divided by the firing frequency with the lights off (FF on/FF off), varied with light intensity, exhibiting significantly higher firing frequency during lights on compared with lights off at intensities of 2 to 3 mW/cm2 or higher. FF on/off for 19 mW/cm2 was 1.62 for 4 to 5 mW/cm2, was 1.51 for 2 to 3 mW/cm2, was 1.39 for 1 to 2 mW/cm2, was 1.18, for 0.6 mW/cm2, was 1.23, and for 0.3 mW/cm2 was 1.1. Light responses to intensities of 19 mW/cm2, 4 to 5 mW/cm2, and 2 to 3 mW/cm2 were significantly different from 1 to 2 mW/cm2 (Fogle, 2011).

The lLNvs anatomically appear to receive input from the compound eyes and the HB eyelet. To determine whether the lLNv light response is due to synaptic inputs from external opsin-based photoreceptors, lLNv were recorded in glass60j (gl60j) mutant flies, which lack all external photoreceptors because of a null mutation in the eyeless gene. The lLNv response to moderate-intensity white light for gl60j flies was 1.37; under intense blue light, the response of gl60j flies was 1.59. Thus, the responses to white and intense blue light do not differ between control and gl60j mutant flies (Fogle, 2011).

The intact lLNv light response in gl60j mutant flies suggests that the blue-light photopigment Cry expressed in the lLNv may underlie the response. Drosophila Cry is excited maximally at 450 nm and absorbs wavelengths no longer than 530 nm, so the lLNv response to discrete wavelength ranges was tested. The lLNvs significantly increased their firing rate in response to both moderate intensity blue-green light (<550 nm) and low-intensity blue-violet light (375 to 450 nm), but not to red-orange light (>550 nm). The spectral profile of the lLNv light response does not differ when tested in gl60j mutant flies. The lack of lLNv responsiveness to wavelengths > 550 nm shows that infrared does not contribute to light-driven increases in firing frequency. The spectral profile of the lLNv light response matches that of Cry but requires about five orders of magnitude higher light intensity than that required for Cry’s known role in resetting the circadian clock via Tim degradation and occur between four to five orders more rapidly than the first biochemical indications of Cry-mediated Tim degradation. Last, unlike Cry-mediated Tim degradation, the lLNv light response is reversible, suggesting that the two Cry-mediated phenomena occur via distinct mechanisms (Fogle, 2011).

Hypomorphic cryb mutant flies, which exhibit impaired circadian function because of a point mutation in the flavin chromophore binding site, display a weakened lLNv response to white light compared with that of their control counterparts. the light response of cryb lLNvs was quantified and its spectral properties was tested. As in control and gl60j flies, white, blue-green, and blue-violet wavelengths evoke a significant increase in firing frequency in cryb mutant flies’ lLNvs. However, these cryb light responses are significantly smaller than for their corresponding wavelengths in control flies (Fogle, 2011).

To determine whether Cry is necessary for the lLNv light response, lLNvs were recorded from two Cry-null lines, cry01 and cry02. Immunocytochemistry confirms baseline native Cry expression in control flies, replicating previous results with this Cry antisera, and no staining of Cry-positive neurons in either Cry null line; however, Cry expression was selectively rescued in the LNvs in both Cry null lines when UAS-Cry was driven by pdfGAL4. The lLNvs of the cry01 and cry02 null flies exhibited normal spontaneous firing but no light response, indicating that Cry is required for the lLNv light response. The lLNv light response was restored to levels indistinguishable from controls by LNv Cry expression in both cry01 and cry02. Although the Pdf driver does target the sLNvs in addition to the lLNvs, light sensitivity was observed in the lLNvs of flies whose sLNvs have been genetically ablated. Thus, it is concluded that the Cry-driven lLNv light response is cell-autonomous and independent of Cry expression in external photoreceptors or other central brain neurons (Fogle, 2011).

To determine whether Cry can optogenetically confer electrophysiological light responsiveness to inherently light-insensitive neurons, Cry expression was targeted to olfactory projection neurons with the GH146-GAL4 driver. This is a rigorous test of (1) Cry's ability to autonomously confer light responsiveness, (2) a conserved mechanism for coupling Cry light activation to membrane changes in nonclock neurons, and (3) independence of Cry/Tim interaction. Cry-expressing olfactory neurons increased firing rate recorded in voltage clamp mode in response to intense blue light, whereas control Cry-minus olfactory neurons were nonresponsive to white, blue-green, blue-violet, intense blue, and orange-red. The spectral profile of the light response of the Cry-expressing (GH146-GAL4/UAS-Cry) olfactory projection cells is almost identical to Cry-positive lLNvs responding to white, blue-green, blue-violet, and intense blue light, but not to orange-red light. The light response of the Cry-expressing olfactory neurons was significantly higher for blue-green, blue-violet, and intense blue light than counterparts measured in control (non-Cry-expressing) cells (Fogle, 2011).

To probe the mechanism of the Cry-mediated light response, the ability of different Cry isoforms expressed in the LNv to rescue the light response in Cry-null flies was assessed. The crym mutant (a nine-amino acid C-terminal truncation of Cry) binds equally well to Tim in light and dark, whereas wild-type Cry interaction with Tim is light-dependent. This allowed asking whether a light-dependent Cry-Tim interaction is necessary for the acute light response of the lLNvs. The firing rate of CryM-expressing lLNv increased significantly in response to white or blue-violet light, but not to red-orange light. Significant large light responses were recorded in the lLNvs in tim-null flies for moderate-intensity white and low-intensity blue-violet light, but not for moderate-intensity orange-red light. Thus, Tim interaction is not necessary for the Cry-mediated lLNv light response (Fogle, 2011).

In addition to having the Drosophila-like light-responsive Cry1, many insects express a second, vertebrate-like Cry2 that is a potent transcriptional regulator but lacks light sensitivity. Because Drosophila Cry may also act as a transcriptional regulato, lLNvs expressing either monarch butterfly (Danaus plexippus) dpCry1 or dpCry2 was recorded in the LNvs of cry01 flies. The lLNvs expressing dpCry1 were electrophysiologically responsive to moderate-intensity white and low-intensity blue-violet light but not to moderate-intensity orange-red light versus white light. In contrast, l-LNvs expressing dpCry2 showed no significant response to white, blue-violet, or orange-red light. Paired t tests comparing firing frequency in light versus dark showed no significant response for any wavelength for dpCry2. Thus, the light response requires Cry that is light-sensitive but not transcriptionally active (Fogle, 2011).

Because a point mutation destabilizes the flavin chromophore binding site in cryb mutants, it was asked whether acute inhibition of the light-activated flavin redox reaction of Cry blocks the lLNv light response. The lLNv respond to blue-violet light at baseline. Within 5 min of bath-applied flavin-specific redox inhibitor diphenyleneiodonium chloride (DPI), the response was attenuated. Within 10 min of DPI exposure, blue-violet light no longer evoked a light response. In contrast, the lLNv light response in vehicle-treated controls was stable over 30 min. Note that DPI does not alter spontaneous baseline firing frequency recorded from the lLNv for up to 30 min, indicating the absence of nonspecific metabolic effects on firing (Fogle, 2011).

Light-activation of the flavin chromophore of Cry appears to couple to depolarization of neuronal membrane potential, resulting in increased firing rate. The amplitude of intense blue light-evoked resting membrane potential changes was tested in current clamp of opsin-free gl60j flies; then the hypothesis was tested that potassium channel modulation underlies coupling of light-activated Cry to membrane depolarization. To visualize light-evoked lLNv membrane potential changes more clearly, action potentials were blocked in these recordings well past saturation with the voltage-gated sodium channel blocker tetrodotoxin (TTX), and the resting membrane potential (RMP) response to light was assessed. TTX did not significantly reduce these shifts at 100 nM, 500 nM, or 1 microM. Light-evoked membrane potential changes were, however, significantly decreased by a subsequent application of a voltage-gated and inward rectifier potassium channel blocker cocktail including 10 mM tetraethylammonium (TEA), 2 mM 4-aminopyridine (4-AP), and 2 mM cesium chloride in the presence of 100 nM TTX. Time-matched controls were performed with 100 nM TTX only, and found that resting membrane changes were found to not differ from control. The K-blocker cocktail also significantly disrupted membrane potential shifts in the absence of TTX. These results suggest that potassium channel modulation couples to the Cry-mediated lLNv light response (Fogle, 2011).

To determine the precise timing of the Cry-mediated light response, recordings were made in lLNv of gl60j mutant flies illuminated with a software-triggered blue light source. After ~60 cycles of 4-s blue light pulses from four different lLNvs, records were analyzed in 100-ms bins for spike frequency, 1 s before and after light onset. Although a trend toward more action potentials can be observed during the light pulse, this measurement does not clearly resolve the onset of increased firing in response to light at a millisecond time scale. However, application of episodic blue light pulse protocol followed by averaging 120 traces (30 each from four lLNvs) precisely registered by light-on and -off effectively filters the noise from individual records and yields a clear light-evoked RMP response. Kinetic analysis reveals that the averaged light-evoked response is best fit with two exponentials with a fast component and a slower component. Similarly, the averaged return to the baseline 'dark' RMP is also best fit with two exponentials. Notably, the fast components of the on and off response are nearly identical. The speed of the on response (~100 ms) is within an order of magnitude of that of classical opsin-based phototransduction, suggesting that coupling light-activated Cry to depolarization may be diffusion-limited and require intermediate steps.

The white light intensity levels used to characterize the lLNv electrophysiological light response, although five times higher than intensities necessary to induce Cry-Tim interaction, correspond to natural light levels typically observed in the early to mid-morning on a clear day, consistent with recent findings that the lLNv are light-activated morning arousal neurons. It was asked what percentage of blue light permeates the cuticle and reaches central brain neurons. That the average transmittance through the head cuticle was 55%. Importantly, the transmittance through the eye cuticle is similar at 57% (Fogle, 2011).

Cry mediates a rapid electrophysiological light response that is distinct from classical opsin-based phototransduction. However, the present results do not rule out the possibility that light-activated synaptic inputs from external photoreceptors co-modulate lLNv firing rate in intact animals. Dye-filled individual lLNv cells show extensive arbors in the optic lobe and may reflect sites for external photoreceptor input. All lines of evidence herein indicate that the Cry-mediated electrophysiological light response is mechanistically distinct from the previously described Cry-Tim interaction. Qualitatively, the Cry-mediated electrophysiological light response bears some resemblance to melanopsin-based light activation of retinal ganglion cells (which underlies circadian entrainment in mammals), whereby light activation leads to increased action potential firing rather than graded potentials found typically in image-forming photoreceptors. The Cry-mediated electrophysiological response appears to exhibit a higher light threshold compared with opsin-based light sensing, which may bias its physiological functions to non-image-forming photosensitive cells (Fogle, 2011).


REGULATION

The clock input to the first optic neuropil of Drosophila melanogaster expressing neuronal circadian plasticity

In the first optic neuropil (lamina) of the fly's visual system, two interneurons, L1 and L2 monopolar cells, and epithelial glial cells show circadian rhythms in morphological plasticity. These rhythms depend on clock gene period (per) and cryptochrome (cry) expression. This study found that rhythms in the lamina of Drosophila may be regulated by circadian clock neurons in the brain since the lamina is invaded by one neurite extending from ventral lateral neurons; the so-called pacemaker neurons. These neurons and the projection to the lamina were visualized by green fluorescent protein (GFP). GFP reporter gene expression was driven by the cry promotor in cry-GAL4/UAS-GFP transgenic lines. It was observed that the neuron projecting to the lamina forms arborizations of varicose fibers in the distal lamina. These varicose fibers do not form synaptic contacts with the lamina cells and are immunoreactive to the antisera raised against a specific region of Schistocerca gregaria ion transport peptide (ITP). ITP released in a paracrine way in the lamina cortex, may regulate the swelling and shrinking rhythms of the lamina monopolar cells and the glia by controlling the transport of ions and fluids across cell membranes at particular times of the day (Damulewicz, 2011).

This study showed a single projection from the pacemaker cells in the brain to the lamina, in which several structural circadian rhythms have been detected. Moreover, this input probably originates from the 5th small LNv. Since the 5th s-LNv does not express PDF, this cell is different from the other LNvs. The possibility that this process originates from other clock cells, for example from the LNds, and extends to the aMe first, and next to the lamina cannot be excluded. A CRY-positive LNd, which is immunoreactive to ITP, could invade the lamina by passing the aMe first. This neuron, however, is also immunoreactive to sNPF, but the projection detected in the lamina is immunoreactive to ITP only. It indicates that this projection originates from the 5th s-LNv, which is immunoreactive to ITP but not to sNPF. This study examined GFP expression driven by cry-GAL4 in thin, 20 microm cryostat sections and thick 100 microm vibratom sections of the Drosophila brain. In most earlier studies on clock neurons and their projections, whole-mount preparations of the Drosophila brain were used, or the lamina was cut-off during preparation. Such procedures from previous studies meant that the very fine projection from the brain to the lamina could not be observed. This study detected the projection by using 20 microm sections and collecting confocal optical sections at a 1 microm interval (Damulewicz, 2011).

In several previous studies, it has been suggested that CRY is present in different types of clock neurons. These results have been obtained using various methods; cry-GAL4 driven GFP expression, cry mRNA in situ hybridization, immunolocalization and cry deletion mutants. Using cry-GAL4 line and 20 microm sections of the D. melanogaster brain, it was found that CRY is located in all s-LNvs, l-LNvs, LNds, DN1s and DN3s but is absent in DN2s and LPNs. These results only partly confirm the results of earlier studies. It has been shown that LNvs but only some DN1, and three or four from the six LNd are CRY - positive, while DN2, DN3 and LPNs are CRY-negative. One study did d not detect CRY in DN2s and DN3s, and in about half of the LNds and DN1, but cry promoter dependent reporter genes and cry mRNA can be detected in these neurons. In this study, all of LNds showed GFP fluorescence in the cry-GAL4 strain, but only 3-4 cells were found to be CRY-immunopositive using antibodies. In turn, using the in situ hybridization method, cry mRNA was not detected in those cells. Since the pattern of cry-GAL4 driven GFP expression depends on the transgene insertion site and whether the first intron of the transgene has been inserted, spatial and circadian regulation of cry was examined. A series of cry-GAL4 transgenes containing different portions of cry upstream and intron 1 sequences was examined. The first intron was shown to drive expression in eyes and antennae, and upstream sequences induce cry expression in brain clock neurons and in peripheral oscillators; in eyes and antennae. In addition, upstream sequences also induce expression of cry, in other non-clock cells in the optic lobe (Damulewicz, 2011).

The results obtained using various methods suggest that in the case of CRY, translation and cry transcription may be specifically regulated. CRY-positive labeling in the 4th LNd was observed in flies kept for 5 days in constant darkness. Flies kept longer in this condition brought on weak staining in one of the DN2 neurons. Thus, the level of CRY in this neuron may be very low, and the CRY level may only be detected after it has accumulated for several days in DD. It is possible, that in some of the LNds, DN1 and DN3 cry expression is very low and protein is undetectable by the immunohistochemistry method, or that cry mRNA is unstable and CRY protein is not synthesized. Among six LNds, three neurons, that show a strong signal of GFP in the brain cryostat sections used in this study, may correspond to CRY-positive cells detected in the studies of other authors. In turn, three LNds with weak GFP in these preparations may correspond to CRY immunonegative cells. These cells had about a 50% lower GFP level than the rest of the LNds at all time points, except at ZT4 when their GFP fluorescence was lower by 20% (Damulewicz, 2011).

Beside neurons, clock genes have also been detected in glial cells. A subpopulation of glial cells in the brain of Drosophila have rhythmic expression of per gene, and they are necessary for maintaining circadian locomotor activity. However, the presence of CRY in glia was not detected in this study. In the optic lobes, GFP driven by cry-GAL4 was observed in many non-clock cells in which the localization pattern was very similar to the distribution of glial cells. But these non-clock cells were not labeled with the antibody against REPO protein, a specific marker for glial cells. The REPO protein is required for glia development and differentiation and has been detected in all types of glia in the adult brain of Drosophila. The analysis of cry-GAL4 driven GFP and REPO immunolabeling showed no co-localization between CRY and REPO. However, in the close vicinity of GFP-positive cells, REPO-positive glial cells were observed. A similar result was obtained using the antibody against the Drosophila vesicular monoamine transporter (DVMAT), which enabled labeling the fenestrated glia in the optic lobe. These results suggest that CRY is present in non-clock neurons in the optic lobe, but not in glial cells (Damulewicz, 2011).

In addition to localization of cry-GAL4 driven GFP in cell bodies of neurons, GFP processes were also detected invading three neuropils in the optic lobe. In the medulla, a dense network of processes originate from DN3s and their terminals seem to form synaptic contacts with not-yet identified target cells. The regular network of processes was also detected in the lobula but their origin is unknown. The most interesting finding is the projection of CRY-positive processes to the lamina. Although the lamina showed robust circadian remodeling of neuron morphology, a circadian input had not been previously detected. In the lamina, per is probably expressed in the epithelial glial cells, however, maintaining the lamina structural rhythms also requires per expression in the retina photoreceptors and in the LNs (Damulewicz, 2011).

Beside PER, CRY is also important for circadian rhythms in the lamina. In an earlier study, it was shown that the circadian rhythm in morphological plasticity of L2 dendritic trees, is not present in per01 mutant while its phase depends on CRY. In cryb mutant, the pattern of daily changes in size of the L2 dendritic tree was different than in wild-type Canton-S flies. In males and females of Canton-S wild-type flies, the largest L2 dendritic tree was found at the beginning of the day. This daily pattern of the structural changes of L2 dendrite resembles the pattern of cry mRNA cycling in Drosophila heads and bodies, and in the 5th s-LNv detected in this study. Although the L2 dendritic tree is the largest at the beginning of the day in the distal lamina, its axon, as well as the axon of L1 monopolar cell, swell at the beginning of both day and night. These changes have been detected in the proximal lamina. Moreover, the α-subunit of the Na+/K+-ATPase and subunits of the V-ATPase also show diurnal changes in abundance in the lamina. Such an occurrence indicates that circadian rhythms in cell structural plasticity are correlated with rhythmic changes in the level of proteins involved in the transport of ions. The rhythm in the α-subunit of the Na+/K+-ATPase level is bimodal with two peaks; in the morning and in the evening. This pattern is changed in the cry0 mutant. It indicates that CRY is not only important for the maintenance of the daily pattern of morphological changes of the L2 dendritic tree but CRY also helps to maintain cycling of the Na+/K+-ATPase in the epithelial glial cells in the lamina (Damulewicz, 2011).

It is uncertain whether there is regulation of lamina rhythms by the brain pacemaker because connections between the pacemaker neurons in the accessory medulla and the lamina have not been observed. It was found, however, that rhythms in axon plasticity of neurons in the lamina are circadian, have two peaks (morning and evening) and are synchronized with locomotor activity. The present results now show, that thin neurite extends from the aMe and arborizes in the distal lamina. In the aMe, the s-LNvs are regarded as the main pacemaker cells maintaining circadian rhythms. The l-LNvs are involved in behavioral arousal and sleep. For these reasons, the LNvs are good candidates as oscillators controlling lamina rhythms. Moreover, all LNvs except the 5th s-LNv, express PDF which may synchronize central oscillators with each other and with peripheral ones. In the housefly, large PDF-immunoreactive neurons, similar to Drosophila's l-LNvs, have terminals in the lamina which show circadian structural changes. Moreover, these neurons cyclically release PDF that affects circadian plasticity in the lamina. In Drosophila, release of PDF from PDF-immunoreactive processes in the medulla, where these processes form a dense network of varicose processes, is also possible. These processes, however, do not extend to the lamina. In the present study, PDF immunolabeling of the newly described Drosophila's CRY-positive terminals in the lamina was negative. This does not exclude PDF action in the lamina, particularly when PDF receptors have been detected in non-neuronal cells between the lamina and the retina. PDF may diffuse in the lamina after release from terminals in the distal medulla (Damulewicz, 2011).

Ion transport peptide (ITP) and short neuropeptide F (sNPF) have been detected in the LNvs. Among the five s-LNvs, ITP was found in the 5th s-LNv, while sNPF was observed in four other s-LNvs which also express PDF. In the present study, ITP-immunoreactive fibers were detected, using the Schgr-ITP antisera, in the distal lamina, co-localized with cry-GAL4 driven GFP. The co-localization with ITP suggests that the projection into the lamina may originate from the 5th s-LNv. Little is known about the function of the 5th s-LNv. It has been suggested, that this neuron, together with LNds and some DN1s, drive the evening peak of D. melanogaster bimodal activity. The finding indicates a possible new function of the 5th s-LNv in regulating circadian structural rhythms in the lamina, since this neuron is immunoreactive to ITP. Like other peptides in the optic lobe, ITP seems to be released from varicose terminals in a paracrine way. This conclusion was reached because no synaptic contacts between ITP-immunoreactive processes and cells in the lamina were detected. This peptide probably diffuses in the distal lamina and may facilitate chloride and/or other ion-dependent swelling and shrinking of the L1 and L2 axons. At least two ion pumps; the V-ATPase and Na+/K+-ATPase, show robust cyclical activity in the epithelial glial cells. The epithelial glial cells swell and shrink in anti-phase to the L1 and L2 interneurons. Preliminary results showed that in a transgenic line carrying RNAi to block ITP expression, the pattern of rhythmic changes in the level of the α-subunit of the Na+/K+-ATPase in the lamina glial cells of Drosophila is different than the pattern in wild-type flies. Thus, not only CRY but also ITP is important for maintaining rhythmic activity changes of the Na+/K+-ATPase (Damulewicz, 2011).

The function of ITP in the nervous system is unknown. In the lamina ITP may play a similar regulatory role as in hindgut of insects, transporting ions and fluids across cell membranes (Damulewicz, 2011).

Since the L1 and L2 monopolar cells swell in the morning and in the evening, ITP released from the 5th s-LNv may drive the evening peak of this rhythm. This is thought to be so, because the 5th s-LNv and LNd are regarded as the lateral neurons' evening oscillator. In turn, PDF may drive the morning peak because PDF is thought to control the morning peak of locomotor activity, in a LD 12:12 regime. However, PDF's role in promoting locomotor activity in the evening has also been shown. The role of ITP as a neurotransmitter of circadian information to the lamina and as a possible regulator of rhythmic swelling and shrinking of the L1 and L2 monopolar cells, requires more experimentation and will be the subject of the next study (Damulewicz, 2011).

Cryptochrome-mediated phototransduction by modulation of the potassium ion channel β-subunit redox sensor

Blue light activation of the photoreceptor Cryptochrome (Cry) evokes rapid depolarization and increased action potential firing in a subset of circadian and arousal neurons in Drosophila melanogaster. This study shows that acute arousal behavioral responses to blue light significantly differ in mutants lacking Cry, as well as mutants with disrupted opsin-based phototransduction. Light-activated Cry couples to membrane depolarization via a well conserved redox sensor of the voltage-gated potassium (K+) channel β-subunit (Kvβ) Hyperkinetic (Hk). The neuronal light response is almost completely absent in hk-/- mutants, but is functionally rescued by genetically targeted neuronal expression of WT Hk, but not by Hk point mutations that disable Hk redox sensor function. Multiple K+ channel α-subunits that coassemble with Hk, including Shaker, Ether-a-go-go, and Ether-a-go-go-related gene, are ion conducting channels for Cry/Hk-coupled light response. Light activation of Cry is transduced to membrane depolarization, increased firing rate, and acute behavioral responses by the Kvβ subunit redox sensor (Fogle, 2015).

Acute behavioral arousal to blue light is significantly attenuated in CRY mutants. This study identified a redox signaling couple between blue light-activated CRY and rapid membrane depolarization via the redox sensor of Kvβ channel subunits coassembled with Kvα channel subunits. Additional unknown factors may act as intermediates between CRY and Hk. This finding provides in vivo validation of a very longstanding hypothesis that the highly conserved redox sensor of Kvβ subunit functionally senses cellular redox events to physiological changes in membrane electrical potential. Genetic loss of any single component functionally disrupts the CRY-mediated blue light response, which is functionally rescued by LNv restricted expression of their WT genes in the null backgrounds. Although little is known about the structural contacts between Kvβ and EAG subunits, Kvβ subunits make extensive physical contacts in a fourfold symmetric fashion in 1:1 stoichiometry with the T1 assembly domain of other coassembled tetrameric Kvα subunits that form the complete functional channel (Fogle, 2015).

Application of Kvβ redox chemical substrate modulates voltage-evoked channel peak current, steady-state current, and inactivation in heterologously expressed α-β channels, which are reversed by fresh NADPH. These results indicate that measurements of channel biophysical properties can reflect the redox enzymatic cycle of Kvβ as these channel modulatory effects are absent in preparations that lack the expression of WT Kvβ subunits or express redox sensor mutant Kvβ subunits. Whether direct chemical redox reactions occur between CRY and Hk is unclear. For CRY, light or chemical reduction induces one-electron reduction of the FAD cofactor of CRY, whereas the reductive catalytic mechanism of AKRs (such as Hk) requires a hydride ion transferred from NADPH to a substrate carbonyl, then a solvent-donated proton reduces the substrate carbonyl to an alcohol. These differences in redox chemistry between CRY and Hk suggest that other intermediates, such as oxygen, are possibly required for redox coupling (Fogle, 2015).

Spectroscopic analysis of animal and plant CRYs suggest that light activation causes reduction of the FAD oxidized base state. Light activation of Drosophila CRY also evokes conformational changes in the C terminus of CRY that clearly promotes CRY C-terminal access to proteolytic degradation and subsequent interactions with the Timeless clock protein, thus signaling degradation and circadian entrainment. However, all existing evidence suggests that light activated CRY-mediated circadian entrainment and membrane electrical phototransduction operate under different mechanisms, including their different activation thresholds and relative dependence on the C terminus of CRY. Further distinguishing the distinct mechanisms of the downstream effects of light-activated CRY, the light-induced conformational changes that couple CRY to ubiquitin ligase binding (thus causing circadian entrainment) occur in oxidized and reduced states of CRY and are unaffected in CRY tryptophan mutants that presumably are responsible for intraprotein electron transfer reactions following light-evoked reduction of the FAD cofactor. Another recent study shows that light- or chemical-evoked reduction of Drosophila CRY FAD is coupled to conformational changes of the CRY C terminus, along with reporting a surprising negative result that DPI has no effect on the reoxidation of the reduced anionic semiquinone of purified Drosophila CRY. DPI could hypothetically influence the electrophysiological light response by blocking the pentose phosphate pathway, which produces the Hk redox cofactor NADPH, but this does not explain the light dependence for DPI blocking the electrophysiological light response herein. The available evidence indicates that CRY-mediated light evoked membrane depolarization occurs independently of conformational changes in the CRY C-terminal domain but depends on redox changes in CRY, whereas CRY-mediated light evoked circadian entrainment depends on conformational changes in the CRY C-terminal domain and may or may not depend on CRY redox state (Fogle, 2015).

Light-activated CRY evokes rapid membrane depolarization through the redox sensor of the Kvβ subunit Hk. A general role for circadian regulation of redox state coupled to membrane excitability has been described recently in mammalian suprachiasmatic neurons. Redox modulation of circadian neural excitability may be a well-conserved feature (Fogle, 2015).

A neural network underlying circadian entrainment and photoperiodic adjustment of sleep and activity in Drosophila

Sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. This study shows that the Hofbauer-Buchner eyelets, located between the retina and the medulla in the fly optic lobes, differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)- and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, it was shown that the large LNvs interact with subsets of 'evening cells' to adjust the timing of the evening peak of activity in a day length-dependent manner. This work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms (Schlichting, 2016).

Circadian clocks create an endogenous sense of time that is used to produce daily rhythms in physiology and behavior. A defining characteristic of a circadian clock is a modest deviation of its endogenous period from the 24.0 h period of daily environmental change. For example, the average human clock has an endogenous period of 24 h and 11 min. Thus, to maintain a consistent phase relationship with the environment, the human clock must be sped up by 11 min every day. A sensitivity of the circadian clock to environmental time cues (zeitgebers) ensures that circadian clocks are adjusted daily to match the period of environmental change. This process, called entrainment, is fundamental to the proper daily timing of behavior and physiology. For most organisms, daily light/dark (LD) cycles are the most salient zeitgeber (Schlichting, 2016).

Although most tissues express molecular circadian clocks in animals, the clock is required in small islands of neural tissue for the presence of sleep/activity rhythms and many other daily rhythms in physiology. Within these islands, a circadian clock neuron network (CCNN) functions as the master circadian clock. Subsets of neurons within the CCNN receive resetting signals from photoreceptors, and physiological connections between these neurons and their clock neuron targets ensure light entrainment of the CCNN as a whole (Schlichting, 2016).

In both mammals and insects, the CCNN receives light input from multiple photoreceptor types. In Drosophila, the CCNN is entrained by photoreceptors in the compound eye, the ocelli, the Hofbauer-Buchner (HB) eyelets, and by subsets of clock neurons that express the blue light photoreceptor Cryptochrome (CRY). Understanding how multiple light input pathways modulate the CCNN to ensure entrainment to the environmental LD cycle is critical for understanding of the circadian system and its dysfunction when exposed to the unnatural light regimens accompanying much of modern life (Schlichting, 2016).

This study investigates the physiological basis and circadian role of a long-suspected circadian light input pathway in Drosophila: the HB eyelets. These simple accessory eyes contain four photoreceptors located at the posterior edges of the compound eyes and project directly to the accessory medullae (AMe), neuropils that support circadian timekeeping in insects. In Drosophila, the AMe contain projections from ventral lateral neurons (LNvs), important components of the CCNN that express the neuropeptide pigment dispersing factor (PDF), an output required for robust circadian rhythms in locomotor activity. The axon terminals of the HB eyelets terminate near PDF-positive LNv projections and analysis of visual system and cry mutants reveals a role for the HB eyelet in the entrainment of locomotor rhythms to LD cycles, but how the eyelets influence the CCNN to support light entrainment is not well understood (Schlichting, 2016).

This study presents evidence that this circadian light input pathway excites the small LNvs (s-LNvs) and acts to phase-dependently advance free-running rhythms in sleep/activity while inhibiting the large LNvs (l-LNvs). This work reveals that input from external photoreceptors differentially affects specific centers within the fly CCNN. Furthermore, it was shown that, under long summer-like days, the l-LNvs act to modulate subsets of so-called evening cells to delay the onset of evening activity. These results reveal a neural network underlying the photoperiodic adjustment of sleep and activity (Schlichting, 2016).

The experiments described in this study lead to two unexpected findings regarding the network properties of circadian entrainment in Drosophila. First, the l-LNvs govern the phase of evening peak of activity through PdfR-dependent effects on evening cells that bypass the s-LNvs. Although previous work has implicated the l-LNvs in the control of evening peak phase, the current results are the first to provide evidence that there is a direct connection between the l-LNvs and evening cells within the AMe and that this connection mediates the photoperiodic adjustment of sleep and activity in the fly. Second, the HB eyelets light input pathways, long implicated in circadian entrainment, have opposing effects on the l-LNvs and s-LNvs, inhibiting the former and exciting the latter. These results reveal not only a differential effect of a light input pathway on specific nodes of the CCNN but also establish that light from extraretinal photoreceptors can have synergistic or antagonistic effects on CRY- and compound eye-mediated light responses, depending on the clock neuron target in question (Schlichting, 2016).

Both the l-LNvs and s-LNvs express the blue light circadian photoreceptor CRY, the expression of which renders neurons directly excitable by light entering the brain through the cuticle. How such CRY-mediated light input interacts with input from external photoreceptors is not well understood, although it is known that each system alone is sufficient for the entrainment of locomotor rhythms. Genetic evidence suggests that the HB eyelets have relatively weak effects on circadian entrainment: flies with functional eyelets that lack compound eyes, ocelli, and CRY entrain relatively poorly to LD cycles relative to flies with functional eyes or CRY. The small phase responses of locomotor rhythms to HB eyelet excitation further supports a relatively weak effect of the eyelet on free-running locomotor rhythms (Schlichting, 2016).

The LNvs are critical nodes in the CCNN and are closely associated with input pathways linking the central brain to external photoreceptors. Work on the LNvs has provided evidence for a division of labor among the l-LNvs and s-LNvs: the l-LNvs are wake-promoting neurons that acutely govern arousal and sleep independently of the s-LNvs, whereas the s-LNvs act as key coordinators of the CCNN to support robust circadian timekeeping. Anatomical and genetic evidence has long supported the notion that the dorsal projections of the s-LNvs represent the key connection between the LNvs and the remaining components of the CCNN. However, a smaller body of work has suggested that the l-LNvs also contribute to the entrainment of sleep/activity rhythms under LD cycles. The Pdf knockdown and PdfR rescue experiments under long day conditions indicate that, as the day grows longer, the l-LNvs play a greater role in the timing of the evening peak. Moreover, the effects of PDF released from the l-LNvs are mediated not by the PDF receptive s-LNvs but rather by the fifth s-LNvs and a subset of the LNds, the NPF and ITP coexpressing LNds in particular (with some influence of the other PDF-receptor positive LNds). These same neurons were recently identified as evening cells that are physiologically responsive to PDF but relatively weakly coupled to LNv clocks under conditions of constant darkness. The results suggest that the l-LNvs differentially modulate the NPF/ITP-positive evening oscillators as a function of day length, producing stronger PDF-dependent delays under long day conditions through increased release of PDF from the l-LNvs, thereby delaying the evening activity peak. Thus, the l-LNvs mediate their effects on the evening peak of activity through their action on the NPF/ITP-positive subset of evening oscillators. The proposed PDF release from the l-LNvs under long days requires their activation via CRY and/or the compound eyes via ACh release from lamina L2 interneurons. It is hypothesized that the inhibitory influence of the HB eyelets ceases under long days allowing the compound eyes and CRY to maximally excite the l-LNvs. Indeed, previous work has established that the compound eyes are especially important for adapting fly evening activity to long days. Furthermore, several studies have suggested that the compound eyes signal to the l-LNvs leading to enhanced PDF release and a slowing-down of the evening oscillators. A recent paper measuring Ca2+ rhythms in the different clock neurons in vivo supports this view (Liang, 2016): Ca2+ rhythms in the l-LNvs peak in the middle of the day, unlike the s-LNvs, which display Ca2+ peaks in the late night/early morning. It is suggested that this phasing is produced by the inhibition of l-LNvs by the eyelets in the morning, followed by the excitation of the l-LNvs by the compound eyes and CRY. Interestingly, the only other clock neuron classes to display Ca2+ increases during the day are the LNds and fifth-sLNv, which phase lag the l-LNvs by ~2.5 h and display peak Ca2+ levels in the late afternoon, a time that coincides with the evening peak of activity (Liang, 2016). It is proposed that the relative coordination of Ca2+ rhythms between the l-LNvs and the LNds/fifth-sLNv is produced by the connection this study has identified between these neurons and the action of the eyelet and visual system on the l-LNvs (Schlichting, 2016).

Recent work has revealed that evening activity is promoted directly by the evening oscillator neurons and that the mid-day siesta is produced by the daily inhibition of evening oscillators by a group of dorsal clock neurons (Guo, 2016). It is proposed that the connections described in this study govern the timing of the evening peak of activity through the PDF-dependent modulation of the molecular clocks within the evening oscillator neurons, although PDF modulation likely results in the excitation of target neurons, which would promote evening activity. The results reveal new and unexpected network properties underlying the entrainment of the circadian clock neuron network to LD cycles. Excitatory effects of light on the LNvs are differentially modulated by the HB eyelets via cholinergic excitation of the s-LNvs and histaminergic inhibition of the l-LNvs. The work further reveals PDF-dependent modulatory connections in the AMe between the l-LNvs and the s-LNvs and, most surprisingly, between the l-LNvs and a small subset of evening oscillators. This work indicates that the latter connection is critical for the adjustment of evening activity phase during long, summer-like days. This network model of entrainment reveals not only how CRY and external photoreceptors interact within specific nodes of the CCNN, but also how photoreception is likely to drive changes in CCNN output in the face of changing day length (Schlichting, 2016).

Transcriptional Regulation

How is Cryptochrome mRNA cycling affected by mutations in four clock genes implicated in gene regulation: per, tim, Clock, and cycle? In all single mutants and double mutant combinations, little or no mRNA cycling is found, indicating that cycling requires a functional pacemaker and is not merely light driven. cry mRNA levels are a function of the specific mutant or mutant combination. They are relatively low in the per or tim null mutants as well as in the per;tim double mutant combination, whereas they are relatively high in the Clock and cycle mutants. The double mutants per;Clock and per;cycle also show high cry mRNA levels, indicating an epistatic effect of Clock and cycle over per. Thus, CRY mRNA levels are low in per and tim null mutants, the opposite of what is observed for autoregulation of PER and TIM mRNA levels. CRY mRNA levels are high in clock or cycle mutants, contrary to the low PER and TIM mRNA levels found in these novel clock mutants (Emery 1998 and references).

A recessive third chromosomal mutation that abolishes bioluminescence rhythms has been identified, cryb. cryb is an apparent null mutation in a gene encoding Drosophila's version of the blue light receptor cryptochrome. To determine the mutation's effects on per and tim transcription, a per-luc or a newly generated tim-luc fusion gene (each encoding luciferase sequences only) were introduced into homozygous mutant genetic backgrounds. luc-reported expression in both cases is arrhythmic. In contrast to other recently identified mutations affecting per and tim expression (Allada, 1998 and Rutila, 1998), the new mutation does not give rise to profound subnormalities in overall levels of per and tim expression in mutant flies. Nevertheless, western blot analyses using head extracts of mutant flies maintained in LD show that the levels of Tim and Per protein remain at high levels throughout the day and night, relative to the very low troughs observed during the daytime in wild type. In addition, Tim and Per proteins are anomalously present in both hypo- and hyperphosphorylated forms in a temporally unchanging manner. That Tim stays at the same levels during the day and night in the mutant is especially interesting, because the rapid disappearance of this protein in response to light is the earliest response to this stimulus of a known component of Drosophila's rhythm system. Yet the absence of rhythmic clock gene transcription indicates that the mutant is doubly defective. This is because either of two regulatory phenomena is sufficient to drive Tim cycling (reviewed by Young, 1998): oscillating tim expression (which occurs in the absence of environmental fluctuations) or light suppression of TIM (in the absence of tim mRNA cycling). Against this background, the absence of effects of some (but not all) orthodox visual mutations on light-induced Tim degradation is notable (Yang, 1998), as is the fact that peak sensitivity for this light effect is in the blue range (Suri, 1998). Thus, the new mutation might uniquely affect elements of the light entrainment pathway, which would include extraocular reception and processing of blue light inputs. Alternatively, the mutation could affect a protease whose targets include Tim and Per (Stanewsky, 1998).

If that is not the case, and the new mutation causes a specific defect in the light entrainment pathway, protein oscillations in temperature cycles should not be affected. Western blots of extracts from mutant and normal heads showed that Per and Tim fluctuated robustly in 12 hr:12 hr, 25°C:20°C cycles; such cyclings continued in constant conditions. The daily mobility shifts of Per and Tim are apparent in both wild type and mutant genetic backgrounds, indicating that the phosphorylation program can function in the mutant (Stanewsky, 1998).

Targets of Activity

Although most circadian clock components are conserved between Drosophila and mammals, the roles assigned to the Cryptochrome (Cry) proteins are very different: Drosophila Cry functions as a circadian photoreceptor, whereas mammalian Cry proteins (mCry1 and 2) are transcriptional repressors essential for molecular clock oscillations. This study demonstrates that Drosophila Cry also functions as a transcriptional repressor. RNA levels of genes directly activated by the transcription factors Clock (Clk) and Cycle (Cyc) are derepressed in cryb mutant eyes. Conversely, while overexpression of Cry and Period (Per) in the eye repressed Clk/Cyc activity, neither Per nor Cry repressed individually. Drosophila Cry also represses Clk/Cyc activity in cell culture. Repression by Cry appears confined to peripheral clocks, since neither cryb mutants nor overexpression of Per and Cry together in pacemaker neurons significantly affected molecular or behavioral rhythms. Increasing Clk/Cyc activity by removing two repressors, Per and Cry, leads to ectopic expression of the timeless clock gene, similar to overexpression of Clk itself. It is concluded that Drosophila Cry functions as a transcriptional repressor required for the oscillation of peripheral circadian clocks and for the correct specification of clock cells (Collins, 2006).

Several pieces of evidence point to Drosophila Cry, like its mammalian counterparts, functioning as a repressor of Clk/Cyc-activated transcription: (1) expression of four Clk/Cyc target genes is derepressed in cryb mutants; (2) overexpression of cry together with per is sufficient to repress tim and vri expression in the eye, and this is supported by Cry repressing Clk/Cyc-activated transcription in transfected cells, either alone or in conjunction with Per; and (3) removing both Cry and Per leads to ectopic tim expression in the brain (Collins, 2006).

Although Cry and Per seem to function together to repress Clk/Cyc activity, the results do not imply a direct interaction between Cry and Per proteins. Drosophila Cry-Per interactions have been detected in yeast, but Cry and Per appear to interact only via Tim in vivo. Furthermore, Per continues to repress Clk/Cyc activity in vivo during the first half of the day, presumably after Cry has been degraded by light. Thus, Cry and Per seem to control distinct steps in repression of Clk/Cyc activity, with Cry probably initiating, and Per maintaining, repression. Further experiments will be required to test whether Tim also facilitates repression. While in vitro studies indicated that Tim helps remove Clk/Cyc from DNA, in vivo studies of the timUL mutant suggests that Tim does not participate in repression of per and tim transcription and instead stabilizes Per and facilitates its nuclear entry. Given that Drosophila Tim interacts with both Per and Cry in vivo, it will be interesting to test whether the Per-Cry interactions detected in mammalian clock cells are mediated via mTim (Collins, 2006).

Very little is known about the developmental specification of clock neurons. Per and Cry normally restrict tim expression to cells that adopt a circadian cell fate. The results complement experiments in which overexpression of Clk led to ectopic tim expression, since they reveal that cells not normally destined to develop as clock cells repress Clk/Cyc activity during development. However, there must be additional factors that contribute to clock cell fate, since the ectopic Tim+ve cells in per01; cryb double mutant larvae did not produce PDP1. Similarly, there must be unidentified factors that maintain repression of tim in nonclock cells, since repression of Clk/Cyc activity will prevent further per expression. The presence of extra Tim-expressing cells may also explain the Tim-dependent rhythmic behavior of per01; cryb in LD cycles, since ectopic Tim expression influences LD behavior (Collins, 2006).

The findings that Cry functions as a repressor in Drosophila are supported by the high conservation across species of the “core” photolyase-like domain of Cry, which is sufficient for repression in Xenopus. TheDrosophila crym mutation removes most of the Cry C terminus and interferes with Cry's response to light. However, CryM still supports a functional clock in the eyes, suggesting that the remaining core of CryM functions as a transcriptional repressor (Collins, 2006).

Cry's homology with DNA photolyases has led to the suggestion that Cry was the original molecule that allowed organisms to respond to light -- primitive organisms could detect light and regulate gene expression with one molecule (Cry) to avoid damage by sunlight during light-sensitive processes such as DNA replication. While ancestral Cry may have acted as both a light sensor and repressor, non-Drosophilid insects such as the monarch butterfly Danaus plexippus have two cry genes and divide repressor/light sensor function between them. Thus, circadian clocks may well have their origins in rapid responses to light, and the anticipatory clock gene networks could have subsequently been built around Cry, a light-responsive protein and a transcriptional repressor, the function of which has gradually become specialized (Collins, 2006).

The cryptochrome (cry) gene and a mating isolation mechanism in tephritid fruit flies

Two sibling species of tephritid fruit fly, Bactrocera tryoni and Bactrocera neohumeralis, are differentiated by their time of mating, which is genetically determined and requires interactions between the endogenous circadian clock and light intensity. The cryptochrome (cry) gene, a light-sensitive component of the circadian clock, was isolated in the two Bactrocera species. The putative amino acid sequence is identical in the two species. In the brain, in situ hybridization shows that cry is expressed in the lateral and dorsal regions of the central brain where Per immunostaining is also observed and in a peripheral cell cluster of the antennal lobes. Levels of cry mRNA were analyzed in whole head, brain, and antennae. In whole head, cry is abundantly and constantly expressed. However, in brain and antennae the transcript cycles in abundance, with higher levels during the day than at night, and cry transcripts are more abundant in the brain and antennae of B. neohumeralis than in that of B. tryoni. Strikingly, these results are duplicated in hybrid lines, generated by rare mating between B. tryoni and B. neohumeralis and then selected on the basis of mating time, suggesting a role for the cry gene in the mating isolation mechanism that differentiates the species (An, 2004).

Identification of novel genes involved in light-dependent CRY degradation through a genome-wide RNAi screen

Circadian clocks regulate many different physiological processes and synchronize these to environmental light:dark cycles. In Drosophila, light is transmitted to the clock by a circadian blue light photoreceptor Cryptochrome (Cry). In response to light, Cry promotes the degradation of the circadian clock protein Timeless (Tim) and then is itself degraded. To identify novel genes involved in circadian entrainment, an unbiased genome-wide screen was performed in Drosophila cells using a sensitive and quantitative assay that measures light-induced degradation of Cry. The expression of ~21,000 genes was systematically knocked down and those that regulate Cry stability were identified. These genes include ubiquitin ligases, signal transduction molecules, and redox molecules. Many of the genes identified in the screen are specific for Cry degradation and do not affect degradation of the Tim protein in response to light, suggesting that, for the most part, these two pathways are distinct. The effect of three candidate genes on Cry stability was further evaluated in vivo by assaying flies mutant for each of these genes. This work identifies a novel regulatory network involved in light-dependent Cry degradation and demonstrates the power of a genome-wide RNAi approach for understanding circadian biology (Sathyanarayanan, 2008).

Of the ubiquitin-conjugating enzymes identified in this screen, a composite E2-E3 ligase, Bruce, and a novel HECT domain-containing E3 ligase CG17735 had the strongest effects on light-dependent Cry degradation. Neither of these ligases had a significant effect on the Tim light response in flies. Bruce exerts its anti-apoptotic role through its N-terminal IAP domain or by targeting proapoptotic proteins, such as SMAC2, for degradation using its C-terminal E2-E3 ligase activity. In flies, apart from its anti-apoptotic activity, it also plays an important role in regulating sperm individualization, a process by which the sperm nuclear syncytium differentiates into individual sperm (Huh, 2004). This is similar to the apoptotic pathway proteins, DIAP1, DIAP2, and Dredd, which perform a nonapoptotic role in the IMD immune pathway. Thus a nonapoptotic role for Bruce in regulating Cry stability is not surprising. It is interesting to note that Bruce knockdown in S2 cells has a small effect on baseline levels but primarily blocks light-induced Cry degradation, suggesting that Cry is rendered stable and nonresponsive to light in the absence of Bruce. The genetic evidence from loss-of-function mutations of Bruce in the fly suggest that Bruce affects the stability of Cry in LD cycles. The same appears to be true for the dual specific phosphatase SSH. This effect on Cry in LD cycles most likely also represents impaired degradation in response to light. It is inferred that the mechanisms involved in the Cry response to parametric and nonparametric entrainment may be somewhat different, and the cell culture assay may allow detection of molecules required for either mechanism. The fact that all these candidates identified in the cell culture screen have the same general effect on Cry stability in cells and flies demonstrates the power of the screen for identifying novel circadian regulatory molecules (Sathyanarayanan, 2008).

CG17735 affects the degradation of Cry by light in cell culture as well as in flies. Given that this gene encodes a putative E3 ligase, it is tempting to speculate that this is the E3 ligase that targets Cry for degradation in response to acute light exposure. However, until a null mutant of CG17735 can be obtained, the extent to which it is required for the regulation of Cry remains unknown. Nor can the behavioral consequences of completely blocking Cry degradation be known. It is intriguing that the knockdown of CG17735 is associated with a long period, but at this point it is unclear if this is due to the effect on Cry or on some other, as yet unidentified, clock target (Sathyanarayanan, 2008).

Although various components of the Cry degradation complex were identified, the importance of light-dependent Cry degradation to circadian resetting behavior is not clear. Bruce mutants demonstrate a strong defect in Cry degradation; however, due to the weak behavioral rhythms in these mutants, the effect on circadian photic sensitivity could not be addressed. Also it is noted that light-dependent Cry degradation, although reduced, still persists in BruceRB mutants, suggesting that it is regulated by redundant pathways. The other two mutants assayed (ssh and CG17735) did not completely abolish Cry degradation either, but in this case, it may be because neither is a null mutant. Interestingly, Tim degradation was not affected in any of these mutants, suggesting that they specifically affect Cry degradation. Tim is targeted for light-dependent degradation by an F-box-containing protein, Jet, which has minimal effect on Cry degradation. This result again supports the existence of two distinct destruction complexes that regulate Cry and Tim degradation. However, some aspects of the degradation pathways may be similar. The demonstration that SkpB, a component of the SCF complex, is critical for both Cry and Tim degradation suggests that some components are shared between the two destruction complexes. In addition, components required for the activation of Cry are presumably also required for the Tim light response. It is likely that other classes of molecules identified in the screen, such as those that affect redox status, are involved in Cry activation and therefore also affect Tim and the behavioral response. Further analysis of these molecules in clock neurons should address the role of redox and Cry degradation in circadian photosensitivity (Sathyanarayanan, 2008).

Effects of light interruption on sleep and viability of Drosophila melanogaster

Light is a very important regulator of the daily sleep rhythm. This study investigated the influence of nocturnal light stimulation on Drosophila sleep. Results showed that total daytime sleep was reduced due to a decrease in daytime sleep episode duration caused by discontinuous light stimulation, but sleep was not strongly impacted at nighttime although the discontinuous light stimulation occurred during the dark phase of the cycle, the scotophase. During a subsequent recovery period without light interruption, the sleep quality of nighttime sleep was improved and of daytime sleep reduced, indicating flies have a persistent response to nocturnal light stimulation. Further studies showed that the discontinuous light stimulation damped the daily rhythm of a circadian light-sensitive protein Cryptochrome both at the mRNA and protein levels, which subsequently caused disappearance of circadian rhythm of the core oscillator timeless and decrease of Timeless protein at nighttime. These data indicate that the nocturnal light interruption plays an important role in sleep through core proteins Cryptochrome and Timeless, Moreover, interruption of sleep further impacted reproduction and viability (Liu, 2014; 25148297).

Protein Interactions

The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila Cryptochrome (Cry), a putative circadian photoreceptor, interacts with the clock protein Timeless (Tim) in a light-dependent manner. Although Tim dimerizes with Period (Per), no association between Cry and Per has previously been revealed, and aspects of the light dependence of the Tim/Cry interaction are still unclear. Behavioral analysis of double mutants of per and cry suggest a genetic interaction between the two loci. To investigate whether this is reflected in a physical interaction, a yeast-two-hybrid system was employed that revealed a dimerization between Per and Cry. This is further supported by a coimmunoprecipitation assay in tissue culture cells. The light-dependent nuclear interactions of Per and Tim with Cry require the C terminus of Cry and may involve a trans-acting repressor. Thus, as in mammals, Drosophila Cry interacts with Per, and, as in plants, the C terminus of Cry is involved in mediating light responses (Rosato, 2001).

At 25°C, perS;cryb flies display predominantly 24 hr cycles in a LD 12:12 regime, although ~40% of the flies also have a minor 19 hr perS component. These two periodic components are not found together in either single mutant (Rosato, 2001).

Locomotor activity was monitored for perS, cryb, and the perS;cryb double mutants at 18°C and 28°C. Single mutant perS or cryb flies entrain to the LD 12:12 regime at both temperatures, showing a 24 hr period and distribution of activity around the times of light/dark transitions. In DD, they free-run with a period of about 24 hr for cryb and a period of about 19 hr for perS, with a modest temperature dependence. In DD conditions, perS;cryb flies behave virtually identically to perS mutants at both temperatures. However, the behavior of the double mutant changes dramatically in LD, in a temperature-dependent manner. At 18°C, all perS;cryb flies show a periodic component of about 24 hr, but about 60% of them also display a minor 19 hr component. At 28°C, 79% of the rhythmic flies display the endogenous 19 hr period as the main rhythmic component. The breakdown of entrainment at 28°C in double mutant flies could reflect a genuine genetic interaction between the cryb and perS mutations. Alternatively, perhaps the limits of entrainment at high temperature are reduced in cryb mutants so that perS;cryb flies might indeed entrain to a T cycle of 20 hr at this temperature (which is closer to the 19 hr endogenous period of perS), whereas cryb individuals (whose endogenous period is ~24 hr) might not. To test this hypothesis, the locomotor activity rhythms of single and double mutant flies was monitored at 28°C under an LD 10:10 regime. Both perS and cryb flies entrain under this condition. However, the double mutants may show some evidence of entrainment during the first two cycles of the new light/dark regime, but any entrainment soon breaks down, and the perS;cryb flies free-run, with their daytime activity advancing by about 90 min on each successive day. Therefore, the entrainment defect at high temperature shown by perS;cryb flies is the product of a specific interaction between the two mutations rather than a defect in the entrainment of cryb alone. In Drosophila, the visual system is involved in the reception of circadian-relevant light information. This system is perfectly functional in the double mutant and is revealed by the startle response that is evident at the transition points from dark to light (and vice versa) at both temperatures. Therefore, perS;cryb flies are able to detect light but are deficient in the transmission of light information to the clock mechanism in a temperature-dependent manner (Rosato, 2001).

The genetic interaction between perS and cryb prompted an investigatation of the possibility of a physical interaction between Per and Cry using a yeast-two-hybrid system. A full-length Cry protein, directly fused to LexA (bait), was challenged with Per(233-685) as prey. This fragment includes the major protein/protein interaction domains described for Per. A fragment of Tim(377-915) that is known to bind to Per and contains the relevant regions for Per/Tim dimerization as prey was also tested. No interactions were observed between LexA-Cry and both Per(233-685) and Tim(377-915) fragments in the dark. Cry has been shown to interact with full-length Tim, but not Per, under constant light. In light, LexA-Cry binds strongly to Per(233-685), but not to Tim(377-915). LexA-Cry was also challenged with full-length Per and Tim, both in darkness and light. No interactions were observed in the dark. Under constant light, only full-length Tim showed evidence of dimerization with LexA-Cry. Three conclusions are drawn from these results: Per and Tim interactions with LexA-Cry are light dependent; the N and/or the C terminus of Tim are required for the association with LexA-Cry, and there is an inconsistency between the results obtained from full-length Per and the fragment Per(233-685). In regard to the latter, the well-established Per/Tim interaction was retested using LexA-Tim bait with Per and Per(233-685) preys in darkness and light. No interactions were observed using full-length Per. Subsequent Western blot analysis has revealed that, in this system, full-length Per is poorly expressed, thereby explaining the lack of interactions in yeast with this construct. Nevertheless, a strong interaction between LexA-Cry and Per(233-685) could be demonstrated. This discrepancy between the current results and contradictory published results must reside in the different yeast-two-hybrid systems employed. Evidence was also found for a Tim-independent Cry/Per complex using coimmunoprecipitation (Rosato, 2001).

Cryptochromes are believed to interact with a signaling factor after light exposure, and evidence has been found in plants for a role of the C-terminal domain in signaling. Since the coimmunoprecipitation result supports the view that the interaction between LexA-Cry and Per(233-685) in yeast reflects a meaningful association between Per and Cry, the power of yeast genetics was exploited to test the regulatory role of the C terminus of Drosophila Cry. Twenty residues were deleted from the Cry C terminus to create CryDelta and it was challenged with Per(233-685) and full-length Tim in darkness and light. An interaction was evident in both conditions, with no obvious difference between them. It has been suggested that LexA-Cryb is unable to interact with Tim in yeast cells because it may have lost its photoresponsiveness. Both LexA-Cryb and LexA-CrybDelta, which are strongly expressed in yeast, are nevertheless unable to interact with Per(233-685) or with Tim. Given the light independence of CryDelta, it is suggested that the D[410]N substitution in Cryb probably confers a gross structural defect to LexA-Cryb, rather than simply affecting its photoreceptor ability (Rosato, 2001).

To further map the interaction between Cry and Per, LexA-CryDelta was challenged with several overlapping Per fragments. It was confirmed that LexA-Tim (377-915) interacts with the PAS A domain (Per[233-390]) and Per(233-685). LexA-CryDelta does not associate with Per(233-390), nor with the PAS A + B region (Per[233-485]), but interacts with the downstream C domain (Per[524-685], which includes the perS site. From these results, it is speculated that Tim and Cry may interact with different regions of the Per protein and, since Cry associates with region(s) of Tim external to the (377-915) fragment, it is hypothesized that Per, Tim, and Cry can be found in the same complex (Rosato, 2001).

LexA-Cry requires light in order to interact with Per(233-685) and Tim. However, it cannot be ruled out a priori that it is the temperature increase, caused by the continuous light exposure, rather than light per se, that triggers Cry's interactions. LexA-Cry was therefore challenged with Per(233-685) and Tim at 37°C in the dark, but no interactions were observed. Furthermore, since LexA-CryDelta does not require light, this variant was used to investigate the effect of temperature on Cry interactions (Rosato, 2001).

Yeast patches were grown on X-gal plates at 30°C and 37°C in parallel. It was noted that at 37°C, the LexA-CryDelta interaction with Per(233-685) is considerably weakened, whereas the control LexA-Tim(377-915)/Per(233-685) dimerization does not show any substantial temperature differences. The same temperature dependence is also observed when LexA-CryDelta is challenged with Tim and Per (524-685) (Rosato, 2001).

To further identify those regions of Cry that could suppress the negative effect of darkness, random Taq-induced mutations were introduced into full-length cry by PCR, and LexA-Cry* mutants were created by in vivo gap repair. The putative LexA-Crys* were challenged with Per(233-685) in the dark. A total of 14 bona fide light-independent mutations were identified that generated a Cry/Per interaction in darkness. The sequencing of these variants shows that all of these light-independent Crys* carry either a translational stop or a frame-shift at their C termini. Some of the mutants have additional amino acid substitutions scattered across the entire sequence, but because of their sporadic nature, it is very unlikely that these missense mutations are contributing to the light-independent phenotype (Rosato, 2001).

The results reported above support the view that the C terminus of Cry is responsible for the light dependence of the interactions with Per and Tim. Perhaps the removal of the C terminus changes Cry conformation to a form that is active in darkness. Alternatively, there could be a carboxy-terminal-bound, light-inhibited nuclear repressor of Cry in yeast. In fact, trans-acting factor in yeast can be mutated to disinhibit nuclear Cry activity in darkness, and currently, attempts are being made to identify the gene(s) involved (Rosato, 2001).

Thus, it has been shown that Cry binds Per in yeast and in a Drosophila cell culture system. As in yeast, the light-dependent activities of Cry in S2 cells have been reported only in the nucleus, where Cry is suggested to undergo a conformational change after light absorption, allowing it to bind to Tim (and now Per). However, Cry coimmunoprecipitates with Tim and Per in the cytoplasm of S2 cells under darkness, suggesting that light is not required to change Cry into its active conformation. Consequently, both in yeast and S2 cells, it is predicted that a nuclear factor may interact with the Cry C terminus in darkness to prevent it from interacting with the two clock proteins. Cry itself is probably not its own repressor, because full-length Cry was tested in a yeast-two-hybrid assay and it does not significantly self-associate in light or dark. However, mutagenesis of the yeast genome has identified two variants that can derepress the Cry/Per interaction in darkness. Isolation of this gene(s), irrespective of its function in yeast, will provide candidates for this nuclear repressor(s), which might have a clock relevant homolog(s) in Drosophila. An analogous situation has been reported in which Saccharomyces cerevisiae casein kinase I, HRR25 (without known clock function in yeast) binds and phosphorylates Per with affinities similar to the Drosophila casein kinase Iepsilon, Doubletime (Dbt). The signaling mechanism of cryptochrome is also mediated through the C terminus in Arabidopsis. A fusion between ß-glucuronidase (GUS) and the C-terminal domain (CCT) of either Cry1 or Cry2 (to create CCT1 and CCT2) mediates a constitutive light response. This means that 'isolated' CCTs display properties in the dark that are strikingly similar to those of light-activated Crys. Within the Cry molecule, the C-terminal domain is repressed under darkness, and light activation might be achieved either by an intramolecular or an intermolecular redox reaction, but the details of the light-induced activation of CCT are not known. In this study, it has been shown that the intermolecular model is the more appropriate to explain observations with Drosophila Cry. Light-induced activation of Cry removes a regulatory molecule, enabling the binding of Per and Tim, although the possibility exists that the regulatory molecule itself, rather than Cry, could act as the primary photopigment. It will be of interest to see if this model also applies to Arapidopsis cryptochromes. The C-terminal domain of Cry thus becomes a focal point for further studies, and it is probably not a coincidence that it is this region of the otherwise evolutionary conserved Cry molecule that is the most variable (Rosato, 2001).

It cannot be unequivocally concluded that the physical interaction revealed between Per and Cry is responsible for the genetic interaction that occurs in perS;cryb mutants at high temperature, even though this was the experiment that led the authors to test a possible Per/Cry dimerization. However, the Per/Cry interaction is temperature-sensitive in yeast, and it is the Per C domain (which includes the site of perS) that dimerizes with Cry, providing further circumstantial evidence that the genetic interaction between Per and Cry may correlate with the physical interaction. Furthermore, it is tempting to speculate that differences in the PerS/Cry physical interaction may be at the heart of reports that the perS mutants are hypersensitive to light and that flies carrying a small deletion (amino acids 515-568) within the Per C domain display short, poorly temperature-compensated rhythms and an altered behavioral response to light pulses. Perhaps a reduction in the strength of the PerS/Cry association, further decreased at 28°C below a critical threshold, might account for the entrainment defect of perS;cryb double mutants at high temperature. Finally, genetic interactions between short-period mutations perS and perT and the arrhythmic mutation dbtar implicate the C domain in the dynamics of Per phosphorylation by DBT. Taken together, these results suggest that the Per C domain may provide a convergence point for both Cry and Dbt, and it is anticipated that future research may disclose a prominent role for Cry in the fly circadian clock (Rosato, 2001).

Oscillations of the period (per) and timeless (tim) gene products are an integral part of the feedback loop that underlies circadian behavioral rhythms in Drosophila melanogaster. Resetting this loop in response to light requires the putative circadian photoreceptor Cryptochrome (Cry). The early events in photic resetting were dissected by determining the mechanisms underlying the Cry response to light and by investigating the relationship between Cry and the light-induced ubiquitination of the Tim protein. In response to light, Cry is degraded by the proteasome through a mechanism that requires electron transport. Various Cry mutant proteins are not degraded, and this suggests that an intramolecular conversion is required for this light response. Light-induced Tim ubiquitination precedes Cry degradation and is increased when electron transport is blocked. Thus, inhibition of electron transport may 'lock' Cry in an active state by preventing signaling required either to degrade Cry or to convert it to an inactive form. High levels of Cry block Tim ubiquitination, suggesting a mechanism by which light-driven changes in Cry could control Tim ubiquitination (Lin, 2001).

The profile of Cry protein expression in light-dark (LD) cycles suggests that the protein is unstable in the presence of light. Light-induced instability of Cry is supported by experiments in which Cry was expressed in S2 cells. Cells were transfected with a pIZ-cry construct, in which Cry is tagged with a V5 epitope, and it was noted that levels of the protein were reduced by light treatment. To identify the mechanisms that degrade Cry, Cry-transfected cells were treated with light in the presence of proteasome inhibitors MG115 and lactacystin. Both these agents were effective in blocking Cry degradation. This effect of light on both Tim and Cry suggests that the action of a ubiquitin/proteasome degradation pathway may be one of the first events in photic resetting in Drosophila (Lin, 2001).

Light-induced ubiquitination of Tim takes place in S2 cells and CRY mRNA is expressed in S2 cells. Consistent with the presence of endogenous Cry in S2 cells, it was found that light-dependent inhibition of Per-Tim feedback activity, which is known to be Cry dependent, occurs to a significant extent in the absence of transfected Cry. The presence of endogenous photic signaling mechanisms in S2 cells provides a system in which the degradation of transfected Cry could be studied regardless of its ability to transduce a photic signal (Lin, 2001).

To determine whether a specific region of Cry mediates its degradation, different Cry mutants were transfected and their responses to light were assayed. Cry-N (amino acids 1 to 423) has the C-terminal 119 amino acids deleted and Cry-C (amino acids 244 to 542) has the N-terminal 243 amino acids deleted. The cryb mutation is a missense mutation within the sequence that encodes the highly conserved flavin-binding region of Drosophila Cry. It corresponds to the original cry mutation isolated through a genetic screen of Drosophila. Treatment with light does not reduce the levels of any of these mutant Cry proteins. For Cry-N the levels are consistently low with and without light treatment, indicating a general instability of the protein. Cry-C is expressed at high levels, and levels of Cryb are equivalent to those of the wild type. However, none of these proteins show a response to light. Since there is no common sequence that is deleted in all these constructs, this indicates either that Cry degradation requires more than one part of the molecule or that the overall conformation of the molecule is important for its recognition by the degradation system. The large deletions in Cry-N and Cry-C may prevent such a conformation change. For Cryb, the mutation is thought to prevent association with flavin, which may be required for a redox-mediated conformation change (Lin, 2001).

Cry signaling in plants requires redox activity and is mediated, at least in part, by the flavin moiety bound to Cry. This is based on the finding that DPI, which inhibits the transport of electrons from reduced flavin, is effective in blocking Cry-mediated photic signaling in Arabidopsis. In addition, flavins participate in electron transport in other systems, the most relevant system being that of the photolyases, which are homologous to Crys and which are known to repair DNA through an electron transfer mechanism (Lin, 2001).

Based on the data implicating electron transport in Arabidopsis Cry signaling, the effect of electron transport inhibitors on degradation of Drosophila Cry were determined. DPI attenuates Cry degradation. Thus, photic signaling by Drosophila Cry involves redox activity, most likely mediated by the flavin (Lin, 2001).

The presence of endogenous Cry in S2 cells supports the idea that the light-dependent Tim ubiquitination is mediated by Cry. To determine if the Tim response to light requires Cry, Tim levels were assayed in light-pulsed and unpulsed cryb flies. While wild-type flies show the characteristic decrease in Tim levels with light treatment, this response is lacking in cryb flies (Lin, 2001).

The S2 cell system was used to determine the relationship between light-induced Cry degradation and Tim ubiquitination and degradation. One possibility considered was that Cry is required for Tim stability. In this model, light-induced Cry degradation would lead to Tim degradation, perhaps by exposing relevant sites on Tim to phosphorylation and ubiquitination events. Although Tim and Cry do not bind each other in the dark in the yeast two-hybrid system, they can be coimmunoprecipitated from S2 cells, suggesting that they are present in the same complex. Thus, removal of Cry in response to light could affect Tim processing. Alternatively, light exposure may lead to some conformational and/or redox changes in Cry that trigger downstream events, including Tim ubiquitination and Cry degradation. To distinguish between these two possibilities, the time course of Tim ubiquitination and that of Cry were examined for degradation in S2 cells. An increase in Tim ubiquitination is detected within 5 min of light exposure, while Cry levels in the same extracts remain unchanged up to the end of a 30-min light pulse. Thus, overall degradation of Cry does not appear to be required for Tim ubiquitination. The possibility that Cry is removed from a complex with Tim cannot be excluded: it is more likely that in response to light, Cry transmits a signal that leads to Tim ubiquitination (Lin, 2001).

No degradation of Tim in S2 cells could be detected in response to light. This may be due, in part, to the HA tag on the ubiquitin, which could interfere with proteasomal digestion. However, other researchers have also noted that Tim is not turned over upon light exposure in S2 cells. Extended incubation (up to 6 h post-Tim induction) of transfected cells results in Tim degradation in both dark- and light-treated cells (Lin, 2001).

Tim ubiquitination was examined in the presence of electron transport inhibitor DPI. Tim ubiquitination is increased by DPI, although Cry degradation is blocked, which is consistent with the idea that Tim ubiquitination does not require degradation of Cry. In fact, the increased Tim ubiquitination is most likely due to the accumulation of activated Cry, effected through a block either in degradation or in the reconversion of Cry to an inactive form (Lin, 2001).

Light-induced Tim ubiquitination in S2 cells is thought to be mediated by endogenous Cry. To test the effects of increasing Cry levels on light-induced Tim ubiquitination, S2 cells were cotransfected with hs-tim, hs-Ub, and different concentrations of either pIZ-cry or hs-cry and Tim ubiquitination was assayed 2 h after light exposure (Lin, 2001).

High concentrations of both hs-cry and pIZ-cry decrease Tim ubiquitination. However, ubiquitination of Tim is enhanced when hs-cry is transfected. pIZ-cry does not increase Tim ubiquitination when transfected at low concentrations, most likely because this plasmid yields higher levels of Cry expression. Taken together these observations indicate that small increases in Cry promote Tim ubiquitination after 2 h of light exposure but that high levels attenuate it. However, even in the presence of high levels of Cry, Tim ubiquitination increases during the first 10 to 15 min of light treatment. The block at later time points in Cry-overexpressing cells is indicative of a deficit in the maintenance of Tim ubiquitination, which may be due to enhanced deactivation of Cry (Lin, 2001).

The data on the differential effects of low and high Cry concentrations are supported by results of Cry overexpression in transgenic flies. Flies that overexpress Cry under control of the tim promoter show enhanced resetting, while those that express Cry under the actin 5c promoter show a reduction of light-induced phase delays. The difference in the phenotypes of these two overexpression strains may lie in the level of overexpression. To determine whether the reduced resetting in the actin 5c line correlates with reduced Tim degradation in response to light, flies carrying a UAS-cry construct were crossed to others carrying an actin 5c promoter-GAL4 transgene and the resulting progeny was assayed for Tim expression. Tim expression was examined at different times of day by Western blotting of adult fly head extracts. In Cry overexpression flies Tim levels are considerably higher than wild-type levels at time points early in the day but equivalent to wild-type levels at all other time points. Thus, the effect is specific for the early part of the day, when Tim is normally turned over in response to light (Lin, 2001).

Degradation of Cry by light invokes analogies with the plant photoreceptors, phytochrome (PHY) and Cry, both of which are degraded in response to light. Thus, it may be a common mechanism to control levels of the photoreceptor and thereby the strength of the photic response. Moreover, as noted here for Cry, PHY is known to be degraded by the proteasome (Lin, 2001).

The role of the proteasome in degradation of both Cry and Tim also underscores similarities with the cell cycle. The cell cycle is characterized by cycling proteins that undergo phosphorylation and subsequent degradation, in many cases by the proteasome. Both Per and Tim are cyclically phosphorylated and phosphorylation plays a role in turnover of both proteins. For Tim, light-induced degradation is effected through an increase in phosphorylation and ubiquitination. Thus, as for the cell cycle, multiple proteins in the circadian cycle are turned over by the ubiquitin-proteasome pathway. However, Per turnover may utilize a different pathway since ubiquitination of Per has not been observed (Lin, 2001).

The mechanisms that lead to Cry degradation in response to light are not clear, but it is hypothesized that a conformation change in Cry is required. A light-induced conformation change is supported by the following lines of evidence: (1) in the yeast two-hybrid system Cry interacts with full-length Tim in the presence of light but not in the dark; (2) sequences that mediate Cry degradation do not appear to map to a unique part of the molecule, suggesting that the tertiary structure is important; (3) the Cryb protein is not degraded by light. All these mutants were tested in the presence of endogenous Cry, and so their ability to signal was dissociated from their degradation. Although the single amino acid mutated in the flavin-binding region in Cryb could play a direct role in the degradation process, it is far more likely that it affects a flavin-mediated conformation change. The fact that Cryb does not associate with Tim in the yeast two-hybrid system is consistent with an inability to undergo a conformation change (Lin, 2001).

Cry has been shown to block Per and Tim autoregulation of their own RNA synthesis in a light-dependent manner in S2 cells. Since Tim degradation is not detectable in S2 cells, it has been suggested that the inhibition of Tim activity by Cry, rather than its degradation, is the primary response to light. This block in Per-Tim activity may be the immediate response of the clock to light. Presumably this block persists as long as the photic signals are present and Cry is not degraded. However, a phase change of several hours, which can be produced with a pulse of <1 min of light, must require an irreversible change in a clock component. Tim is ubiquitinated in S2 cells within 5 min of light treatment. In flies, Tim degradation (which presumably follows ubiquitination) occurs within 30 to 60 min of light treatment and is apparently critical for resetting the clock. A Cry molecule with a functional flavin-binding domain is required for this response (Lin, 2001).

Signaling by flavins frequently involves a redox change. In fact, a reagent that blocks the transfer of electrons from reduced flavin prevents Cry degradation by light. At the same time, it increases Tim ubiquitination. Based on the recently proposed models for Arabidopsis Cry, DPI may block either intramolecular electron transport required for a change in Cry conformation or intermolecular transport to a signaling pathway that effects degradation. Assuming that active Cry, which promotes Tim ubiquitination, is produced by a conformation change, it is suggested that the DPI-sensitive step occurs after the conformation change. It should be noted that DPI can also block the activity of other flavoproteins, such as NADPH oxidase and nitric oxide synthase, that play a role in redox processes (Lin, 2001).

Cryptochromes (CRYs) are flavoproteins important for the molecular clocks of animals. The Drosophila cryptochrome (Cry) is a circadian photoreceptor, whereas mouse cryptochromes (mCRY1 and mCRY2) are essential negative elements of circadian clock transcriptional feedback loops. The Drosophila circadian clock involves an autoregulatory feedback loop, in which Period (Per) and Timeless (Tim) inhibit their own transcription by associating with the transcriptional activators Clock (Clk) and Cycle (Cyc). Light affects the feedback loop by causing rapid degradation of Tim by the proteasome. Drosophila cryptochrome appears to mediate this light effect, as suggested by its binding to Tim and Per in yeast and in Drosophila Schneider 2 (S2) cells, its own light-dependent degradation by the proteasome in S2 cells, and its light-dependent ability to inhibit Per:Tim transcriptional repression in S2 cells. It has been proposed that reduction/oxidation (redox) reactions are important for Cry light responsiveness and mCRY1 transcriptional inhibition. Therefore the role of redox in light-dependent activation of Cry and in mCRY1 transcriptional inhibition was evaluated in Drosophila Schneider 2 cells. Using site-directed mutagenesis, three of the four conserved flavin binding residues in dCRY were found to be essential for light responses, whereas three of the four corresponding residues in mCRY1 did not abolish transcriptional responses. Two tryptophan residues in dCRY are critical for its function and are likely involved in an intramolecular redox reaction. The corresponding tryptophan residues do not play a redox-mediated role in mCRY1 function. The data provide a multistep redox model for the light-dependent activities of Cry and suggest that such a model does not apply to mCRY1 transcriptional responses (Froy, 2002).

Intramolecular redox is critical for Cry functions. Photolyases repair UV light-induced pyrimidine dimers through intermolecular redox between the reduced flavin, FADH-, and DNA. However, in bacterial photolyases, an intramolecular redox pathway has also been discovered. Under purification conditions or oxidative stress, the flavin molecule is oxidized into FAD- and becomes catalytically inert. Upon irradiation with white light, the photolyase regains its activity by transferring to the flavin an electron through an internal chain of three tryptophans. To study whether electron transfer through the corresponding tryptophans might be involved in Drosophila Cry function, these tryptophan residues were mutated to alanine in Cry (W342A, W397A, and W420A) and the activity of the mutant proteins was evaluated in S2 cells, using the transcriptional assay and a proteolysis assay (Froy, 2002).

Cry-W342A and Cry-W397A are deficient in mediating light-induced transcriptional derepression, showing no significant light responsiveness , whereas Cry-W420A was significantly light responsive and was not significantly different from wild-type Cry in this response. Similarly, Cry-W342A and Cry-W397A are not light responsive in the proteolysis assay, whereas Cry-W420A was consistently degraded by light. Even though Cry-W342A lost its light responsiveness and, in most assays, proved to be inactive, in some assays, this mutant had limited ability to relieve Per:Tim inhibition, independent of the lighting conditions. The residual activity could be due to the intact W397 that may still be able to transfer electrons to the flavin, but at very low efficiency (Froy, 2002).

Because Cry-W397A was poorly expressed, another mutant, Cry-W397L, was generated to determine whether the loss of activity in Cry-W397A was due to its low level of expression or an inability to mediate redox. Leucine was selected for this substitution because it is a small hydrophobic residue, like alanine, and is unlikely to participate in redox-mediated reactions. Cry-W397L lost its light responsiveness and activity, while retaining a high level of expression (Froy, 2002).

To determine whether the loss of activity of Cry-W342A and Cry-W397A/L was secondary to a structural change, or whether their inactivity was due to a block in intramolecular redox, each of these tryptophan residues were replaced with tyrosine or phenylalanine, residues that are structurally unlike tryptophan but are still capable of electron transfer. Importantly, the light-dependent activity was fully restored by all four mutations (W342Y, W342F, W397Y, W397F) in both the transcriptional assay and the proteolysis assay. These results strongly suggest that intramolecular redox is involved in the light-induced activity of Cry in cell culture and that W420 is less critical than the other two tryptophans for this redox pathway. Furthermore, generation of a double mutant, Cry-W342A-R381A, suggests that flavin excitation is accompanied by intramolecular redox for Cry activation (Froy, 2002).

Serotonin modulates circadian entrainment in Drosophila

Entrainment of the Drosophila circadian clock to light involves the light-induced degradation of the clock protein timeless (Tim). This entrainment mechanism is inhibited by serotonin, acting through the Drosophila serotonin receptor 1B (5-HT1B). 5-HT1B is expressed in clock neurons, and alterations of its levels affect molecular and behavioral responses of the clock to light. Effects of 5-HT1B are synergistic with a mutation in the circadian photoreceptor cryptochrome (Cry) and are mediated by Shaggy (Sgg), Drosophila glycogen synthase kinase 3beta (GSK3beta), which phosphorylates Tim. Levels of serotonin are decreased in flies maintained in extended constant darkness, suggesting that modulation of the clock by serotonin may vary under different environmental conditions. These data identify a molecular connection between serotonin signaling and the central clock component Tim and suggest a homeostatic mechanism for the regulation of circadian photosensitivity in Drosophila (Yuan,2005).

Serotonin regulates the entrainment of circadian behavioral rhythms in Drosophila by affecting the molecular response to light. By modulating the expression of the 5-HT1B receptor in clock neurons, a role of this receptor subtype has been established in the regulation of Drosophila circadian photosensitivity. The data also demonstrate that the molecular connection between 5-HT1B signaling and the clock is GSK3β, which directly phosphorylates the central clock component Tim. It is proposed that serotonin signaling is a part of the homeostatic regulation that prevents dramatic fluctuations in the phase of the circadian clock. In addition, given the altered levels of serotonin in extended DD, it may confer selectivity on the response of the clock to light under different environmental conditions (Yuan, 2005).

The expression pattern of 5-HT1B, as determined by both UAS-Gal4 experiments and by immunostaining, provides some clues to its functions in Drosophila. Besides LNvs and SE5HT-IR neurons, major compartments of the fly brain that express the 5-HT1B receptor include the optic lobes, PI neurons, and mushroom bodies. Interestingly, expression in each of these locations is consistent with functions proposed for serotonin signaling in other organisms. In the housefly, the neuropil of the optic lobes undergoes daily structural changes regulated possibly by serotonin and PDF. PI neurons are neurosecretory cells that may also participate in the ocellar phototransduction pathway. The mushroom body is important for olfactory learning and memory in Drosophila. Therefore, in addition to its postsynaptic function in the LNvs, 5-HT1B may be involved in other aspects of physiology and behavior (Yuan, 2005).

The effect of 5-HT1B on Tim was especially pronounced in the small LNvs. One of the differences between the large and small LNvs is in the timing of nuclear entry, which is delayed in the small subgroup. If delayed nuclear entry accounts for the increased resistance of Tim to light in the small LNvs, it would suggest that 5-HT1B signaling largely affects cytoplasmic Tim (Yuan, 2005).

In addition to its effect on the light response, 5-HT1B overexpression influences free-running behavioral rhythms of cryb flies. It is speculated that this is due to the loss of synchrony among LNs. The mutual coupling of oscillators within an organism is important for the generation and synchronization of circadian rhythms, and serotonin is implicated in this process in some insects. Decreased synchrony may also result from the reduced photosensitivity produced by 5-HT1B overexpression. Interestingly, a significant number of glass, cryb double mutants, which lack CRY as well as all visual photoreceptors, are arrhythmic in DD (Yuan, 2005).

5-HT1B not only affects circadian photosensitivity when over- or under-expressed, it also appears to be the major receptor subtype required for the inhibitory effects of serotonin on entrainment. Notably, when 5-HT1B was knocked down with the RNAi transgene driven by tim-Gal4, the effect on photosensitivity was not as pronounced as with the 5-HT1B-Gal4 driver. This might be due to some background differences in flies carrying the tim-Gal4 transgene, or to nonspecific effects produced by expressing the RNAi construct in irrelevant cells. Also, the possibility that cells other than clock neurons participate in the regulation of light sensitivity via 5-HT1B cannot be excluded. However, clock cells clearly have a major role in this effect, in particular since the circadian response to serotonin is eliminated in the tim-Gal4/RNAi flies (Yuan, 2005).

Effects of serotonin on circadian photosensitivity have been demonstrated in other systems, but the underlying mechanisms were not identified. These studies in Drosophila address this issue by demonstrating an effect of 5-HT1B signaling on the posttranslational modification of Tim via Sgg. In 5-HT1B-overexpressing flies, Tim phosphorylation is reduced, and its stability is increased. In contrast, Sgg phosphorylation is increased (i.e., its activity is decreased) in response to elevated levels of 5-HT1B as well as in response to serotonin treatment. Consistent with this effect of 5-HT1B on Sgg, increased Sgg activity abolishes effects of 5-HT1B overexpression on circadian photosensitivity, while 5-HT1B attenuates the period shortening produced by excess Sgg activity. These reciprocal effects in genetic experiments strongly support the regulation of Sgg activity by 5-HT1B. Expression data indicate that Sgg is expressed predominantly in the cytoplasm. The regulation of cytoplasmic Sgg by 5-HT1B is predicted to affect the phosphorylation status of Tim mainly in the cytoplasm; Sgg-phosphorylated Tim is transported to the nucleus more effectively and is also a better substrate for light-induced degradation (Yuan, 2005).

5-HT1B alone does not significantly affect circadian period, suggesting that its effects on Sgg are limited. In this context, it is noted that, while sgg hypomorphs have a period of ~26 hr, flies hemizygous for the locus have wild-type periods. It is inferred that small (up to 50%) changes in Sgg activity do not alter circadian period but can affect circadian photosensitivity. A role for Sgg in circadian photosensitivity was previously suggested by Martinek (2001) who found that forms of Tim phosphorylated by Sgg were selectively degraded in response to light. In fact, phosphorylated Tim is known to be more sensitive to light. While Sgg appears to be the primary kinase that increases photic sensitivity of Tim, the actual process of light-induced Tim degradation involves the activity of a tyrosine kinase (Yuan, 2005).

These results provide a new mechanism for circadian regulation by a G protein-coupled signaling pathway. A role for GSK3β in the mammalian circadian system was recently reported (Iwahana, 2004). In addition, the mammalian 5-HT1A receptor affects phosphorylation of GSK3β in the mouse brain. It is possible that inhibition of GSK3β activity is a conserved mechanism in the regulation of circadian entrainment in mammals and insects (Yuan, 2005).

Slow dark adaptation has been described in Drosophila, whereby circadian sensitivity to light increases more than 10-fold over 3 days in DD. Increased light responsiveness during dark adaptation occurs in rodents, but the mechanism underlying these effects has not been addressed. Elevated responsiveness to light after prolonged exposure to darkness could be due either to a gain in sensitivity in the sensory system or to an increase in sensory output, which may be caused by a reduction in an inhibitory mechanism. In this study, lower serotonin levels were observed in flies maintained in DD. Given that serotonin signaling modulates circadian light sensitivity, it may be the reduction in this inhibitory mechanism that at least partially accounts for the enhanced light response in prolonged DD (Yuan, 2005).

It is proposed that serotonin signaling, which is itself upregulated by light, is a part of a homeostatic mechanism that regulates circadian light sensitivity. A recent study using human subjects also suggested that serotonin levels in the brain reflect the duration of prior light exposure. This change in serotonin levels with light may be relevant to the etiology and treatment of seasonal affective disorder (SAD), a mood disorder related to the reduced hours of sunlight in winter, particularly at northern latitudes. SAD patients respond to antidepression drug treatments, as well as to light therapy, both of which may produce an increase in serotonin. The interplay of serotonin, light, and the circadian system suggests a close relationship between circadian regulation and mental fitness (Yuan, 2005).

Serotonin modulates the entrainment of the circadian system. In contrast, the current results, and studies done in mammalian systems also, suggest circadian effects on serotonin signaling. (1) Based upon the differences seen in LD versus DD in the fly brain, the level of serotonin is affected by the environmental light cycle. (2) Receptor levels are modulated by circadian components, since 5-HT1B levels are altered in fly circadian mutants. In addition, serotonin release and receptor activity are regulated in a circadian fashion in mammals. Mutual regulation of the circadian and serotonin systems may be necessary to maintain the normal physiological functions of both systems (Yuan, 2005).

The Drosophila circadian network is a seasonal timer

Work in Drosophila has defined two populations of circadian brain neurons, morning cells (M-cells) and evening cells (E-cells), both of which keep circadian time and regulate morning and evening activity, respectively. It has long been speculated that a multiple oscillator circadian network in animals underlies the behavioral and physiological pattern variability caused by seasonal fluctuations of photoperiod. This study manipulated separately the circadian photoentrainment pathway within E- and M-cells and shows that E-cells process light information and function as master clocks in the presence of light. M-cells in contrast need darkness to cycle autonomously and dominate the network. The results indicate that the network switches control between these two centers as a function of photoperiod. Together with the different entraining properties of the two clock centers, the results suggest that the functional organization of the network underlies the behavioral adjustment to variations in daylength and season (Stoleru, 2007).

Two populations of circadian brain neurons, morning cells (M-cells) and evening cells (E-cells), have been connected to morning and evening locomotor activity, respectively (Grima, 2004; Stoleru, 2004). Interactions between the two oscillator populations were studied by selectively overexpressing sgg to speed up the clock in only one cell population or the other (Stoleru, 2005). This study has found that sgg overexpression gives rise to LL rhythmicity, which led to a search for the cellular substrates of entrainment. The rhythmicity is predominantly due to sgg overexpression in E-cells, which suggested that this subset of the clock network is particularly important in the light and that Sgg affects the biochemical pathway through which light impacts clock molecules and adjusts phase to the correct time of day. Indeed, strong evidence is presented that Sgg modulates Cry function, which affects in turn the core clock proteins Per and Tim. The separate manipulation of the Sgg/Cry pathway within E- and M-cells also reveals that the E-clocks drive the behavioral rhythm in light, with prominent Per oscillations of nuclear localization. This light dependence of E-cells contrasts with M-cells, which need darkness to cycle autonomously and dominate the activity output pathway. This distinction suggests a simple dual-oscillator model for how the clock adjusts to photoperiod changes, and support for this seasonal model was obtained by examining E- and M-cell cooperation under different photoperiods (Stoleru, 2007).

The free-running pacemaker and entrainment are two important and increasingly understood aspects of circadian rhythms. In contrast, little information exists about seasonal adjustment, namely, how a constant ~24-hr timekeeper accommodates dramatically different photoperiods. This study shows that the previously defined dual oscillator system in Drosophila, M-cells and E-cells, creates different rhythmic patterns by alternating master clock roles. This understanding emerged from restricting Sgg overexpression to E-cells, which allowed the E-oscillator to function and render flies rhythmic in LL. Sgg probably modulates Cry activity and, when overexpressed, provides sufficient Per and Tim to allow E-oscillator function under constant illumination conditions. The E-clocks therefore manifest free-running properties and function as the master pacemakers in LL, analogous to a previous finding that the M-oscillator is the master in DD (Stoleru, 2005). Nonetheless, these constant conditions, and even the perfect standard LD cycles commonly used in the laboratory, are poor approximations of the changing LD environments found in nature. Circadian oscillators and their entrainment mechanisms have adapted to the dramatic seasonal changes in photoperiod. The previous strategy of using oscillators with different speeds, combined with different photoperiods, has led to a model of alternating control between the M-oscillator and E-oscillator (Stoleru, 2007).

Sgg appears to attenuate, rather than inactivate, Cry activity in E-cells. This is because the LL period of timSgg/PdfGAL80 (~23.5 hr) is longer than the intrinsic period of Sgg-expressing E-clocks in DD (~21 hr) (Stoleru, 2005). A longer period in light is compatible with attenuated light perception under high light intensity conditions (1600 lx, which renders wild-type flies completely arrhythmic) and the application of Aschoff's rule to insects [Aschoff, 1979; One of the earliest observations in the study of circadian rhythms was that continuous light (LL) lengthens circadian period in most nocturnal animal species. 'Aschoff's Rule' posits that there is a positive log-linear relationship between the LL intensity and period]. As there is also a prominent effect on Cry stability, Sgg may be the regulator previously predicted to bind to the Cry C terminus (Busza, 2004; Dissel, 2004). Although Cry is favored as the major circadian substrate of Sgg, there may be others, e.g., the serotonin receptor. Biochemical support for GSK3 involvement in mammalian rhythms has recently been obtained (Yin, 2006). Since GSK3 is a proposed therapeutic target of lithium, the relationship between Sgg and Cry reported in this study recalls the intriguing relationship between mood disorders, light sensitivity, and circadian rhythms (Stoleru, 2007).

The cryb genotype markedly affects DD period in some of the rhythmic genotypes described in this study. Although Cry is probably unnecessary for M-cell rhythmicity, this could reflect some redundancy or assay insensitivity. Moreover, the DD period of cryb is slightly shorter than that of wild-type (23.7 versus 24.4), suggesting that 'dark Cry' makes some contribution to pacemaker function in M-cells as well as E-cells. For these reasons, it is suggested that Drosophila Cry is closer to the central pacemaker than previously believed, and therefore closer to the level of importance of its mammalian paralogs in influencing free-running pacemaker activity. Unlike mammalian Cry, however, Drosophila Cry still appears to function predominantly at a posttranslational level. Indeed, the effects of cryb on Sgg overexpression in DD suggest that the proposed effect of Sgg on Tim stability is really an effect of Sgg on Cry followed by an altered Cry-Tim interaction. It is noted that there is a recent proposal (Collins, 2006) that Drosophila Cry, like mammalian Cry, also functions as a transcription factor in peripheral clocks (Stoleru, 2007).

The importance of E-cells in LL rhythmicity is underscored by the staining results of timSgg/PdfGAL80 brains. Only some E-cells and DN2s manifest robust cycling. It has been suspected that E-cells are important in light because they can rescue the output of arrhythmic M-cells in LD, but not in DD (Stoleru, 2004). Indeed, all of these observations make it attractive to view E-cells as autonomous pacemakers. There is, however, evidence that M-cells may not be completely dispensable. Moreover, a synchronizing or stabilization function is compatible with previous observations under different conditions (Stoleru, 2007).

In the timSgg/PdfGAL80 genotype, only Per nuclear localization changes were detectable near the end of LL cycle. The nature of the assay makes it hard to conclude that there were no differences in total Per staining intensity, i.e., no oscillations in Per levels, so the unique nature of the Per nuclear localization cycling is a tentative conclusion. The same caveat applies to the absence of Tim oscillations and nuclear staining, i.e., negative results cannot exclude low-amplitude oscillations; it is noted, however, that Tim cytoplasmic sequestration has been previously observed in cryb flies after several days in LL. Furthermore, the circadian nuclear accumulation of Tim has been shown to respond differently than that of Per to changes in photoperiod. Nonetheless, Tim could be shuttling with a predominant steady-state cytoplasmic localization, nuclear Tim could be rapidly degraded to create a low nuclear pool, or both (Stoleru, 2007).

The importance of E-cells in entrainment is strongly supported by the potent effect of restricted Cry rescue of cryb: E-cell rescue is much more impressive than M-cell rescue. Moreover, the differences between the two rescued phase response curves (PRCs) are striking; E-cell rescue is virtually complete, whereas the M-cell rescue is notably deficient in the delay zone. In addition, flies with Sgg overexpression in E-cells show altered PRCs, whereas flies with Sgg overexpression in M-cells respond normally to light. The results are strikingly different in darkness, as M-cell-restricted expression causes the typical short period determined by Sgg overexpression, whereas E-cell overexpression has no systemic effect (Stoleru, 2007).

The PRC delay zone is the region impacted most strongly by E-cell Sgg overexpression, indicating that the lights-off early night region is most important to E-cell function and light entrainment. Exposure to light in this interval should mimic long days (summer), which, it is speculated, will delay phase by many hours so that “evening” output of the following day will coincide with the objective evening of the environment. Even the short nights of summer are probably enough time for E-clocks to accumulate sufficient Tim and Per, shuttle them into the nucleus, and reconstitute the rhythmic substrate observed in the Sgg-overexpressing brains in LL. In contrast, M-cells need darkness to cycle robustly. They will become the master clocks and drive the system whenever lights fail to turn on more than 12 hr past lights-off, i.e., during the long nights of winter that mimic the beginning of a DD cycle. Since the intrinsic pacemaker program of M-cells in darkness relies on the changing nature of clock proteins during the night, it is hypothesized that the activity phases under long nights (winter) are locked to lights-off. This suggestion is supported by preliminary data and previous observations showing that per transcription remains locked to lights-off under different entrainment regimes. M-cells are also capable of fully entraining the system in the PRC interval that determines a phase advance (late night). This is consistent with their predicted role in generating an advanced evening output, coincident with the early evenings typical of winter. Otherwise put, long summer days should underlie light primacy as well as long and prominent evening delay zones; both suggest E-cell dominance. Night primacy and M-cells should dominate under winter conditions. This concept endows E- and M-cells with the properties originally envisioned by the Pittendrigh and Daan (1976) dual-oscillator model of entrainment (Stoleru, 2007).

A novel photoreaction mechanism for the circadian blue light photoreceptor Drosophila cryptochrome

Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. An expression and purification scheme was establised that enables purification of mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, this study shows that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FADox) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FADox to the red anionic FAD- radical. These findings lead to the proposal of a novel photoreaction mechanism for dCRY, in which FADox corresponds to the ground state, whereas the FAD- radical represents the light-activated state that mediates resetting of the Drosophila circadian clock (Berndt, 2007; full text of article).

A molecular basis for natural selection at the timeless locus in Drosophila melanogaster

Diapause is a protective response to unfavorable environments that results in a suspension of insect development and is most often associated with the onset of winter. The ls-tim mutation in the Drosophila clock gene timeless has spread in Europe over the past 10,000 years, possibly because it enhances diapause. The mutant allele attenuates the photosensitivity of the circadian clock and causes decreased dimerization of the mutant Timeless protein isoform to Cryptochrome, the circadian photoreceptor. This interaction results in a more stable Timeless product. These findings reveal a molecular link between diapause and circadian photoreception (Sandrelli, 2007).

Wild European populations of Drosophila melanogaster have two major alleles of the timeless (tim) gene, ls-tim and s-tim. These alleles differ in their use of two alternative translational starts to generate longer (L-TIM1421) and/or shorter (S-TIM1398) isoforms. The ls-tim allele is derived from the s-tim allele, and directional selection is thought to have created a latitudinal gradient of ls-tim frequency within the past 10,000 years, perhaps due to an enhanced fitness of ls-tim individuals in temperate environments. TIM is a cardinal component of the circadian clock, and its light sensitivity via its physical interaction with the circadian photoreceptor cryptochrome (CRY) mediates the fly's circadian responses to light. This photoresponse can be quantified at the behavioral level by studying the fly's locomotor response to brief light pulses delivered at zeitgeber time 15 (ZT15), three hours into the night phase of a light/dark [12 hours of light alternating with 12 hours of darkness (LD12:12)] cycle that generates a phase delay of a few hours; the same light stimulus administered late at night (ZT21) generates a phase advance (Sandrelli, 2007).

The enhanced stability of L-TIM might be expected to contribute to the higher levels of TIM observed in natural ls-tim flies and to reduced circadian photoresponsiveness. Circadian light responses in Drosophila are mediated both by the canonical visual pathway, which uses rhodopsins, and by CRY. After stimulation by light, CRY can physically interact with TIM and/or PERIOD in yeast, in Drosophila S2 cells, and in vivo. These PER/TIM/CRY interactions lead to TIM degradation and subsequent PER instability, which releases the negative autoregulation of PER on the per and tim genes. Therefore the physical interaction of the L-TIM and S-TIM isoforms with CRY was examined in the yeast two-hybrid system. No interactions between TIM and CRY occurred in the dark, and the level of interaction between CRY and L-TIM in light was weaker than that between CRY and S-TIM in both plate and liquid assays. As a control, the interaction was examined of L-TIM and S-TIM with the large fragment of PER (residues 233 to 685) that is stable in yeast, but these PER/TIM interactions were not significantly different. These results indicate that the differences in interaction between the two TIM isoforms and CRY are a specific effect due to the additional N-terminal 23 residues in L-TIM, which interfere with the light-dependent dimerization of CRY (Sandrelli, 2007).

A reduced L-TIM/CRY interaction may explain the differences in the fly's circadian photoresponsiveness and the enhanced L-TIM stability. The observation that ls-tim females are more prone to ovarian diapause at any day length (Tauber, 2007) is also consistent with the results presented in this study. As in the corresponding diapause profiles (Tauber, 2007), the transformants conclusively reveal that the circadian photoresponsive phenotypes of natural tim variants are not due to linkage disequilibrium between tim and a nearby locus, but they are attributable to tim itself. Furthermore, the similarity in behavior of natural s-tim variants and P[S-TIM] transformants suggests that the residual putative truncated N-terminal 19-residue TIM product from the s-tim allele does not play any major role in the studied phenotypes (Sandrelli, 2007).

It has been argued that the light sensitivity of the circadian clock needs to be abated in temperate zones because of the dramatic increase in summer day lengths in northern latitudes. One mechanism for this process involves a reduced sensitivity to light-induced disturbance by having a higher pacemaker amplitude. However, the amplitude of TIM cycling in DD was not significantly different between the two variants, nor were there any significant differences in amplitude or phase of the tim mRNA cycle between the s-tim and ls-tim genotypes. Another way to attenuate circadian photoresponsiveness in temperate zones may be by filtering light input into the clock. The molecular changes to the L-TIM protein may buffer the circadian response to light in ls-tim individuals, even in the presence of S-TIM, and may contribute to the positive Darwinian selection observed for ls-tim in the European seasonal environment (Sandrelli, 2007).

Light-dependent interactions between the Drosophila circadian clock factors Cryptochrome, Jetlag, and Timeless

Circadian clocks regulate daily fluctuations of many physiological and behavioral aspects in life. They are synchronized with the environment via light or temperature cycles. Natural fluctuations of the day length (photoperiod) and temperature necessitate a daily reset of the circadian clock on the molecular level. In Drosophila, the blue-light photoreceptor Cryptochrome (Cry) mediates a rapid light-dependent degradation of the clock protein Timeless (Tim) via the F box protein Jetlag (Jet) and the proteasome, which initiates the resetting of the molecular clock. Cry is also degraded in the light but whereas the degradation of Tim is well characterized, the mechanism for light-dependent degradation of Cry is mostly unknown. Until now it was believed that these two degradation pathways are distinct. This study revealed that Jetlag also interacts with Cry in a light-dependent manner. After illumination, Jetlag induces massive degradation of Cry, which can be prevented in vitro and in vivo by adding Tim as an antagonist. The affinity of Tim for Cry and Jetlag determines the sequential order of Tim and Cry degradation and thus reveal an intimate connection between the light-dependent degradation of these two proteins by the same proteasomal pathway (Peschel, 2009).

Jetlag is involved in the resetting mechanism of the circadian clock. Jet is a putative component of a Skp1/Cullin/F-Box (SCF) E3 ubiquitin ligase complex that associates with Tim in a light-dependent fashion in an embryonic Drosophila cell line (S2) in the presence of Cry. This interaction promotes the ubiquitination and degradation of Tim in cultured cells. In nature, two Drosophila allelic variants of timeless can be found: one allele produces a 23 amino acid N-terminally shortened and more light-sensitive form of Tim (s-tim), the other allele encodes both forms (ls-tim). At the molecular level, S-Tim's enhanced light sensitivity is correlated with (and likely due to) enhanced binding to the circadian blue-light photoreceptor Cry (Peschel, 2009).

The hypomorphic jetc mutation carries a single amino acid change in the leucine-rich repeat (LRR) region of Jet, which causes flies to be rhythmic in constant light (LL), but only if they express the less light-sensitive L-Tim protein (encoded by the ls-tim allele) as is the case in Veela flies (Peschel, 2006). The LL-rhythmic Veela phenotype resembles that of cry mutants. Also similar to cryb mutants, homozygous mutant Veela flies accumulate abnormally high levels of Tim protein in the light. Strikingly, both phenotypes are also observed in transheterozygous Veela/+; cryb/+ flies (Peschel, 2006). This strong genetic interaction between tim, jet, and cry prompted an investigation of a potential physical association between Jetlag and Cry proteins in the yeast two-hybrid system (Y2H). In addition, the two different Timeless isoforms were also tested for interaction with Jetlag or Cryptochrome. In agreement with an earlier study, light-dependent interaction between both Tim proteins and Cry was observed, whereby S-Tim interacted more strongly with Cry as compared to L-Tim. Surprisingly, a striking light-dependent interaction between Cry and Jet was observed, but not between Tim and Jet. Given that Tim and Jet do interact in S2 cells cotransfected with cry and the finding that Jet interacts with Cry in yeast, an explanation for the lack of Tim:Jet binding could be that Cry is essential for this interaction (Peschel, 2009).

The interaction between Jetc and Cry is significantly weaker compared with the wild-type protein. Keeping in mind that the LRR is the binding region for the F box proteins' substrate, this weaker association was expected. Additionally, Jet and Jetc were challenged with different Cry mutations. In CryΔ the last 20 residues from the C terminus are missing, resulting in strong, light-independent interactions of CryΔ with Tim. A strong light-independent interaction was observed between Jet or Jetc and CryΔ. The Cryb protein does not interact with Jet or Jetc, correlating with its inability to bind to Tim in yeast (Peschel, 2009).

The strong biochemical and genetic interaction between cry and jet suggests that the Jet:Cry interaction is important in vivo and perhaps required for efficient light-induced Tim turnover. Given that a direct interaction between Jet and Tim was not detected in yeast, this implies that binding of Cry to Tim could modify Tim in a way that it now can bind Jet to induce degradation. Alternatively, the Jet:Cry complex binds to Tim (via Cry acting as a bridge), thereby inducing Tim degradation (Peschel, 2009).

To distinguish between these two possibilities, CoIP experiments were performed in an embryonic Drosophila cell line (S2). A full-length Jetlag protein fused to a HIS-tag (Jet-H) and untagged versions of Cry and Tim proteins were overexpressed in S2 cells and immunoprecipitated with HIS antibody. Cells were grown in darkness and exposed to light for 15 min before performing the assay. As expected from the Y2H results, Cry also interacts with Jet-H in S2 cells. Contrary to the Y2H results, Tim also interacts with Jet-H, without the addition of Cry. When Tim and Cry were simultaneously expressed in the presence of Jet-H, only minimal amounts of Tim protein could be detected in the input or CoIP fractions. It is speculated that the low Tim levels were caused because a fully functional light-sensitive clock-resetting protein complex was reconstituted. To test this, the CoIP experiments were also conducted in the presence of the proteasomal inhibitor MG-132, which led to an overall stabilization of the proteins and a clear demonstration of Tim:Jet interactions in S2 cells. The interaction of Tim with Jet is increased in the presence of Cry, supporting the idea that Cry:Tim or Jet:Cry complexes promote binding of Tim to Jet (Peschel, 2009).

Why could Tim:Jet interactions be detected in S2 cells but not in yeast? The reason for this could be that a crucial phosphorylation step necessary for the detection of Tim by Jet is not performed in yeast, but does occur in Drosophila cells. Alternatively, the low endogenous Cry levels in these cells could promote the Tim:Jet interaction, perhaps contributing to the required posttranslational modification of Tim. Therefore attempts were made to reduce the low endogenous CRY levels even further by cry-dsRNA-mediated interference before conducting the CoIP experiments. dsRNA treatment efficiently reduces transfected Cry levels, indicating that endogenous Cry levels should also be reduced by this treatment. CoIP experiments in the presence of MG-132 show that Jet:Tim interactions are dramatically reduced (but still detectable) after dsRNA treatment. This demonstrates that endogenous Cry levels are supporting Jet:Tim interaction observed in S2 cells (Peschel, 2009).

In cells transfected only with tim, a Jet:Tim interaction was detected, but not a Jet:Cry interaction. Even though the input levels of endogenous Cry and transfected Tim are very low, one would expect to precipitate equal amounts of both proteins bound to Jet, if Cry would indeed form a bridge between Tim and Jet. This was not observed, and only Tim was repeatedly precipitated, indicating the existence of Tim:Jet complexes that are free of Cry. The results therefore support a model in which Cry modifies Tim, allowing Tim to interact with Jet after dissociation of the Cry:Tim complex (Peschel, 2009).

If the Jet:Cry interaction is biologically relevant, an effect on Cry degradation should be detectable in flies with reduced jet function. Indeed, jetc flies exhibited mildly increased Cry levels after 2 and 11 hr in light. Interestingly, in the light phase, s-tim animals accumulate higher levels of Cry compared to ls-tim flies, both in jet+ and jetc genetic backgrounds. Cry associates stronger with S-Tim compared to L-Tim, and in flies this probably leads to a more efficient light-dependent degradation of S-Tim. This suggests that the affinity of the Cry:Tim interaction dictates the temporal order of Tim and Cry degradation by Jet -- in other words, S-Tim would be preferentially degraded, followed by the turnover of Cry, whereas L-Tim enhances the degradation of Cry because of its lower affinity to this photoreceptor (Peschel, 2009).

Because the differences in Cry degradation caused by jetc were subtle, it was desirable to confirm this effect by creating a more severe reduction of Jet function. For this, the stronger jetr allele was combined with jetc or a deficiency of the jet locus. Both combinations lead to substantially increased Cry levels compared to controls and homozygous jetc mutants. This unequivocally demonstrates that jet influences Cry stability in flies. It was also noticed that the absence or presence of eye pigments influences the amount of Cry degradation after light exposure, perhaps because the pigments 'protect' Cry from the light (Peschel, 2009).

Although cry is expressed in S2 cells, the endogenous Cry protein is unstable in S2 cells. jet (but not tim) is also expressed in these cells. Endogenous jet expression may explain the previous observation of Tim ubiquitination in S2 cells without cotransfection of Jet. This suggests that the amount of Jet (and) or Cry is limiting for triggering Tim degradation. To test this, S2 cells were first transformed with cry, jet, and s-tim or l-tim. Cells transfected with cry and tim showed little degradation of Tim, regardless of the Tim form present. In contrast, cotransfection of jet led to massive Tim degradation, suggesting that the endogenous Jet amount is limiting. A slight reduction of Tim degradation was also observed after cotransfection of jetc and the long isoform of Tim, confirming previous results obtained in adult flies (Peschel, 2009).

After establishing conditions that recapitulate light-induced degradation of Tim in cell culture, it became possible to study Cry levels after illumination. Transformation of increasing amounts of jet plasmid DNA is correlated with increased degradation of Cry. This effect is indeed caused by extra jet, because transformation with equal amounts of unrelated plasmid DNA did not result in reduced Cry levels. When the jetc mutation was used, Cry degradation in the light was reduced but still visible, confirming the results obtained with adult flies. Both effects are possibly caused by the poorer ability of Jetc to physically interact with Cry (Peschel, 2009).

So far, these results suggest that Tim is preferentially degraded, when both Tim and Cry are present. If true, addition of Tim should stabilize Cry in S2 cells. Indeed, after 10 or 120 min of light exposure, a dramatic 'protection' of Cry by Tim was observed. Cotransfection of Jet restored the light-induced degradation of CRY, at least after 2 hr of light exposure. It is concluded that Tim indeed protects Cry from light-induced degradation, most likely because it is the preferred target of Jet (Peschel, 2009).

To further prove that both proteins are a target of Jet and subsequent proteasomal degradation, the proteasome inhibitor MG-132 was added to cells transfected with Cry and Jet. As previously shown for Tim, light- and Jet-dependent degradation of Cry was largely prevented after adding the drug, suggesting that Tim and Cry are degraded via the same pathway. Similar as in flies, a minor Jet-dependent reduction of Cry levels, which seems independent of light and the proteasome, was observed, indicating that Jet also promotes Cry degradation via a different, light-independent pathway (Peschel, 2009).

An assay was developed that allowed examination of the light-induced degradation of Cry with a higher temporal resolution and in a more quantifiable manner. A constitutively expressed firefly-luciferase cDNA was fused to full-length Cry (Luc-dCry) or to a C-terminal truncated version of Cry (Luc-dCry528). In S2 cells, the fusion protein Luc-dCry is degraded in a similar way as Cry alone, whereas the truncated Luc-dCry528 is expressed at a very low level. After transient transfection of the luc-dCry gene, luminescence was measured in an automated luminescence counter. After illumination, the Luc-dCry protein is swiftly degraded and in darkness Luc-dCry levels recover, demonstrating that the system nicely reflects the light-dependent degradation of Cry. When Jet is added to the cells the fusion protein is degraded even faster -- an effect not observed when Jetc is added. Lower Luc-dCry levels are observed in the dark portion of the day when Jet is present. Cotransfecting luc-dCry with timeless results in a striking stabilization of Cry in S2 cells, confirming the western blot results. The magnitude of this effect depends both on the isoform and on the total amount of Tim. S-Tim inhibits Luc-dCry degradation more strongly as compared to L-Tim, indicating again that the high-affinity S-Tim:Cry interaction stabilizes Cry more efficiently. Adding Jetlag and Tim at the same time leads to decreased Cry turnover, compared to transfection with Jet alone, but Cry is less protected if Tim is added alone. Overall, these luciferase results nicely confirm the S2-cell and whole-fly western blot results and demonstrate that Jet promotes Cry degradation, which is counteracted by Tim, and especially S-Tim (Peschel, 2009).

Next the Luc-dCry protein was expressed in UAS-luc-dCry transgenic flies under the control of a tim-Gal4 driver. Robust Luc-dCry oscillations, which are due to light-dependent degradation in transgenic flies, was observed, because a sharp decrease of luciferase signals coincides exactly with 'lights-on' in every cycle, and the oscillations immediately stop after transfer to DD. This result is in agreement with light- but not clock-regulated oscillation of the Cry protein in flies. Overexpression of Tim with a UAS-tim transgene led to significantly elevated levels of Luc-dCry during the light phase, which is quite remarkable given that these flies contain the endogenous wild-type allele of jet. Because both transgenic genotypes contained the identical and single copy of the UAS-luc-dCry transgene, this difference in the level of Luc-dCry must be due to the overexpression of Tim. Therefore, the increased daytime Cry levels in the transgenic flies are most likely caused by a stabilization of Cry by Tim, similar to that observed in S2 cells. Also similar as in S2 cells, although the Luc-dCry protein is stabilized by Tim, it can still be degraded by light as long as Jet is present. Interestingly, closer inspection of luc-dCry expression in flies reveals that Cry levels in the UAS-tim flies already recover during the light phase, indicating that Tim mainly protects Cry when light is present. A western blot from flies with the same UAS-tim transgene under the control of a tim-Gal4 driver also reveals a dramatic increase in the levels of Cry and confirms the luciferase results. Both the western blot and real-time luminescence data show that Jet supports the light-dependent degradation of Cry in vitro and in flies and that Tim interferes with this process (Peschel, 2009).

The fact that Tim stabilizes Cry can most easily be explained if Tim is the preferred target for Jet. If true, one would predict that in flies light-induced degradation of both Tim and Cry occurs in sequential order; Tim being degraded ahead of Cry. Therefore Tim and Cry levels were simultaneously measured in head extracts of wild-type flies (y w; s-tim) during the first 10 hr of light in a LD cycle. Although levels of both proteins start to decrease after the lights are turned on, and trough levels are reached at the same time (ZT4), Tim degradation appears to occur more rapid in the early day. This result is in agreement with the idea that Tim is preferentially degraded after initial light exposure. Interestingly, a similar result was reported for Cry and Tim degradation kinetics in adult clock neurons (Peschel, 2009).

Recently, a genome-wide cell-culture-based RNAi screen has been performed in order to identify genes involved in the light-dependent degradation of Cry. Interestingly, Jet was not among the identified candidates. Instead, two other ubiquitin ligases encoded by Bruce and CG17735 were reported to affect light-dependent degradation in flies. The effects reported in this study were caused by eye-color differences between mutants and controls. Therefore, Bruce and CG17735 likely do not contribute to light-dependent Cry degradation in flies, which is also the case for two other ubiquitin ligases that were shown to affect Cry degradation in vitro (Peschel, 2009).

In conclusion, in Drosophila, the clock factor Timeless is degraded after illumination, resulting in a daily reset and adaptation of the circadian clock to its environment. This study has demonstrated that the blue-light photoreceptor Cryptochrome directly interacts with the F box protein Jetlag in a light-dependent manner. This interaction leads to the degradation of Cry by the proteasome and it was unequivocally shown that Jet regulates Cry turnover in vitro and in flies. This is an important and surprising observation, given that so far it was assumed that Cry and Tim are degraded via different pathways. In agreement with previous studies, it was also found that Tim also associates with Jet, but the results suggest that a posttranslational modification of Tim, induced by its binding to Cry, is a prerequisite for the Jet:Tim association. Cry is dramatically stabilized in the presence of Tim, which can be explained by an increased binding affinity of Jet toward light-activated Tim compared to Cry. Based on the results, a more complex model for light resetting is proposed: light induces a conformational change in Cry, allowing it to bind to Tim. S-Tim binds to Cry with higher affinity compared to L-Tim, which leads to more efficient S-Tim degradation by Jet and stabilization of Cry. L-Tim interacts weaker with Cry, presumably resulting in a weaker Jet-L-Tim interaction (or fewer Jet-L-Tim complexes) and less efficient L-Tim degradation. As a result, Cry is less stable in L-Tim flies, because it becomes a better substrate for Jet. Consequently, even less Cry is available to bind to L-Tim, which could further contribute to the reduced light-resetting responses observed in ls-tim flies compared to s-tim flies (Peschel, 2009).

What could be the advantage of such an interdependent binding and degradation of light-regulated clock proteins? The results suggest that Tim and Cry may be degraded in a sequential order. As long as Jet triggers the degradation of Tim, Cry would be spared, presumably because Jet's affinity to light-activated Tim is much higher than to Cry. After Tim levels have decreased to a critical amount, Cry is no longer needed and is now the prime target of Jet. Possibly the degradation of Cry then allows a reaccumulation of Tim in the next circadian cycle, which would also explain why Tim levels start to increase already during the late day (Peschel, 2009).

Flavin reduction activates Drosophila cryptochrome

Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation (Vaidya, 2013).


DEVELOPMENTAL BIOLOGY

Photic entrainment of insect circadian rhythms can occur through either extraretinal (brain) or retinal photoreceptors, which mediate, respectively, sensitivity to blue light and longer wavelengths. Although visual transduction processes are well understood in the insect retina, almost nothing is known about the extraretinal blue light photoreceptor of insects. A candidate blue light photoreceptor gene in Drosophila has been identified, DCry: it is homologous to the cryptochrome (Cry) genes of mammals and plants. The DCry gene is located in region 91F of the third chromosome, an interval that does not contain other genes required for circadian rhythmicity. The protein encoded by DCry is approximately 50% identical to the CRY1 and CRY2 proteins recently discovered in mammalian species. As expected for an extraretinal photoreceptor mediating circadian entrainment, DCry mRNA is expressed within the adult brain and can be detected within body tissues. Indeed, tissue in situ hybridization demonstrates prominent expression in cells of the lateral brain, which are close to or coincident with the Drosophila clock neurons. Interestingly, DCry mRNA abundance oscillates in a circadian manner in Drosophila head RNA extracts, and the temporal phasing of the rhythm is similar to that documented for the mouse Cry1 mRNA, which is expressed in clock tissues. Changes in DCry gene dosage are associated predictably with alterations of the blue light resetting response for the circadian rhythm of adult locomotor activity (Egan, 1999).

RNase protection methods were used to examine various developmental stages and tissues for DCry expression. To determine whether the DCry mRNA is expressed in the brain, RNA samples were prepared from hand-dissected adult brains, which were completely devoid of eyes and ocelli. DCry mRNA can be detected in a modest amount of total RNA, indicating that the message is enriched in the brain. This result also demonstrates that the message is expressed in an extraretinal manner and suggests that it encodes the cryptochrome mediating circadian photoreception. Consistent with expression in the brain, DCry mRNA is detected readily in head tissues of eyes absent (eya) mutants, which entirely lack compound eyes. Interestingly, DCry mRNA can be detected in body tissues, which have been shown to contain photoreceptive clocks, although the relative abundance of the mRNA is apparently lower in the body. Finally, DCry message cannot be detected in moderate-to-large amounts of total RNA from 0-24 hr embryos, whole third instar larvae, or third instar larval brains, suggesting that a different photoreceptor might mediate circadian resetting at these developmental stages. In situ hybridization techniques were used to examine the spatial localization of DCry mRNA within the adult nervous system. A low level of expression can be detected throughout the cell body layer of the CNS. A much stronger signal, however, is observed in large cells of the lateral CNS, which are close to or coincident with the ventral group of Drosophila clock neurons. Specific expression also is detected in adult non-neural tissues, including the gut. Importantly, sections hybridized with a DCry sense probe do not show any signal within brain or gut cells. A small amount of reaction product is observed within the retina (R) with both the sense and antisense DCry probes; thus, it is concluded there is no specific signal for DCry mRNA within retinal tissues (Egan, 1999).

As the mouse Cry1 mRNA had been reported to oscillate in abundance during the diurnal cycle, it was determined whether the same might be true of the DCry mRNA. The DCry message is shown to be more abundant in head RNA samples during the day than at night. In two independent experiments, DCry mRNA was 6- and 11-fold more abundant at peak during the day than it was at the trough of the rhythm during the night. Indeed, the amplitude of the DCry rhythm is greater than that observed for the mouse Cry1 mRNA, which oscillates in abundance in the suprachiasmatic nuclei (SCN). Additional experiments show that DCry mRNA does not show immediate increases in abundance in response to the lights-on signal, indicating that DCry gene expression is not light-inducible. Similar to mouse Cry1 mRNA, the rhythm in DCry abundance persists in constant conditions, demonstrating that it is under circadian regulation (Egan, 1999).

Behavioral genetic experiments were conducted to test the notion that the Drosophila cryptochrome mediates blue light resetting of the circadian clock. As a prelude to these experiments, blue light resetting was examined in normal flies. Normal individuals exhibited phase shifts of increasing magnitude in response to ~200 lux blue light pulses of increasing duration. Flies receiving 5 min of blue light or 5 min of 2500 lux white light exhibit phase delays of identical magnitude, suggesting that this duration of blue light constitutes a saturating light pulse. Importantly, these data indicate that 10 sec and 1 min pulses of blue light cause submaximal phase shifts, and thus such resetting pulses might be appropriate for detecting behavioral alterations that result from changes in DCry gene dosage. To determine whether changing DCry dosage affects blue light resetting, the resetting responses of flies carrying one or two doses of the gene were characterized. Flies heterozygous for Df(3R)Dl-BX12 have significantly smaller phase delays than normal siblings in response to a 10 sec pulse of blue light. Phase delays for such flies are progressively larger in response to longer pulses, but not significantly different from those of siblings, presumably because the system is at or near saturation at the longer pulse durations. These data indicate that flies deficient for DCry product have decrements in blue light resetting (Egan, 1999).

The circadian clock, light, and cryptochrome regulate feeding and metabolism in Drosophila

Recent studies in mammals have demonstrated a central role for the circadian clock in maintaining metabolic homeostasis. In spite of these advances, however, little is known about how these complex pathways are coordinated. This study shows that fundamental aspects of the circadian control of metabolism are conserved in Drosophila. Feeding behavior and basic metabolite levels were assayed in individual flies, and it was shown that, like mammals, Drosophila display a rapid increase in circulating sugar following a meal, which is subsequently stored in the form of glycogen. These daily rhythms in carbohydrate levels are disrupted in clock mutants, demonstrating a critical role for the circadian clock in the postprandial response to feeding. Basic metabolite levels are coordinated in a clock-dependent manner, and clock function is required to maintain lipid homeostasis. By examining feeding behavior, it was shown that flies feed primarily during the first 4 hours of the day and that light suppresses a late day feeding bout through the cryptochrome photoreceptor. These studies demonstrate that central aspects of feeding and metabolism are dependent on the circadian clock in Drosophila. The work also uncovers novel roles for light and cryptochrome on both feeding behavior and metabolism (Seay, 2011).

The circadian clock and metabolic control are dependent upon one another for accurate anticipation of daily changes and physiological optimization for organismal fitness. This study has shown that the Drosophila circadian clock regulates feeding behavior, carbohydrate uptake and storage, lipid homeostasis, and metabolite coordination. The studies also revealed an unexpected role for light exposure and CRY on feeding behavior and metabolism (Seay, 2011).

The rapid and transient increase in circulating sugar concentrations following a meal is one of the best characterized links between nutrition and metabolic homeostasis. These studies have revealed that this classic postprandial response is conserved through evolution in Drosophila. A subsequent >2-fold increase in glycogen concentration was observed, suggesting that circulating sugar is rapidly converted into stored energy. Arrhythmic flies do not display detectable periodicity in their levels of trehalose and glycogen under LD conditions, even though their feeding behavior is similar to wild-type. This indicates that the postprandial increases in circulating and stored carbohydrates are dependent upon a functional circadian clock. Similarly, no significant increases in trehalose or glycogen Triacylglyceride are seen in response to the late day feeding bout in either wild-type flies or cry01 mutants, demonstrating a lack of coordination between feeding and metabolism under these conditions. This loss of coupling between feeding behavior and metabolism has been reported in mammalian systems and is likely due to a disruption of the interactions between central and peripheral oscillators. Interestingly, measurements of glycogen levels in the livers of rabbits, rats, mice, and chickens have revealed daily approximately 2-fold increases with circadian periodicity, similar to the response we observe in flies. Moreover, clock disruption specifically in mouse pancreatic β cells leads to hyperglycemia and diabetes without affecting activity or feeding rhythms. Taken together, these studies define the circadian clock as a central regulator of the normal postprandial response to dietary sugar uptake and the subsequent transient storage of this energy in the form of glycogen (Seay, 2011).

Although the complex biochemistry of carbohydrate metabolism makes it difficult to predict which gene or genes are responsible for the observed oscillation in glycogen levels, 2 candidates are promising. Shaggy is an ortholog of glycogen synthase kinase-3 (GSK-3), which inhibits glycogen synthesis by phosphorylating and inactivating glycogen synthase (Bourouis et al., 1990). Shaggy also regulates the speed of the circadian clock by phosphorylating Timeless protein and controlling its nuclear localization (Martinek et al., 2001). The direct connection of Shaggy with both clock function and glycogen homeostasis suggests that it may provide communication between these 2 systems. A second candidate, glycogen synthase 2, appears to be directly regulated by CLOCK in the rat liver (Doi et al., 2010), although its clock regulation remains unstudied in Drosophila (Seay, 2011).

The approach of analyzing metabolite levels in individual animals also provided a unique opportunity to assess their coordination under LD and DD conditions and in different mutant backgrounds. Highly significant pairwise correlations were found between trehalose, glycogen, and triacylglycerol levels in wild-type flies under LD conditions, reflecting the ability of these animals to maintain normal metabolic homeostasis. These correlations, however, are all reduced under DD conditions. They are also reduced in tim01 mutants under LD conditions, demonstrating that the critical role for the circadian clock in maintaining homeostasis is conserved through evolution. Glycogen and trehalose coordination, however, appears distinct in several ways. First, it is the only metabolite comparison that displays an increased correlation in tim01 mutants when comparing LD and DD conditions. Second, even though wild-type flies in DD display a decrease in their glycogen and trehalose correlation, these metabolites remain highly coordinated in cry01 mutants. The high level of correlation between glycogen and triacylglycerol is also worth noting, given that glycogen is found predominantly in the muscle while triacylglycerol is primarily in the fat body, suggesting that the tissue-specific reserves of stored energy are coordinated by the circadian clock. It will be interesting in future studies to address the roles of peripheral clocks in these pathways (Seay, 2011).

Cry is the primary molecule responsible for entraining the circadian clock to light and is necessary for clock function in some peripheral tissues. Although cry transcripts are most abundant during the day, Cry protein accumulates during the night, likely due to light-sensitive degradation. Short pulses of light can either advance or delay behavior rhythms depending upon when they are administered and Cry activity. Cry is thought to exert these effects through light-dependent degradation of Timeless protein, specifically within the oscillator neurons. Currently, Tim is the only known target of Cry, although Cry has recently been shown to have neuronal functions independent of the circadian clock. The current studies have revealed new roles for Cry in the control of both feeding behavior and metabolism (Seay, 2011).

Late evening feeding is suppressed under normal LD conditions by light signaling through Cry. Thus, animals lacking Cry feed in the evening similar to wild-type flies in DD. This result is unexpected because Cry functions are thought to be restricted to the early morning, when its protein levels are most abundant. In addition, Timeless protein does not provide a likely mechanism to explain the ability of Cry to suppress late day feeding because it is undetectable at this time. This is consistent with the absence of late evening feeding in tim01 mutants under LD conditions. Rather, it is proposed that Cry regulates feeding behavior through a distinct mechanism that remains to be identified (Seay, 2011).

Metabolic processes are also dependent on cry function, likely independent of the circadian clock. Animals lacking Cry can still be entrained to light cycles and retain wild-type circadian rhythms in locomotor activity, eclosion, and molecular oscillations of clock components (Seay, 2011).

In spite of a functioning clock, however, a significant change was found in the phase of glycogen accumulation in cry01 flies. This indicates that Cry delays the accumulation and utilization of glycogen independent of traditional clock function. It is likely that peripheral tissues regulate this oscillation, and it is possible that Cry plays a timekeeping rather than time-setting role in this response, as it may in olfactory neurons. It will be interesting to determine the tissue specificity of Cry function to better understand its roles in regulating feeding behavior and carbohydrate homeostasis (Seay, 2011).

This study shows that Cry, light signaling, and clock function play central roles in controlling feeding behavior and metabolic homeostasis. Adult flies are capable of interpreting seasonal changes in photoperiod and respond with striking alterations in both the frequency and timing of feeding bouts. In addition, fundamental mechanisms of metabolic control, such as the postprandial response and metabolite correlations, are dependent on light signaling and clock function. These studies provide further evidence that key aspects of metabolic control are conserved between Drosophila and mammals and provide a foundation for exploiting the strengths of this model organism toward defining the mechanisms by which this regulation is achieved (Seay, 2011).

Human cryptochrome-1 confers light independent biological activity in transgenic Drosophila correlated with flavin radical stability

Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. This study shows by developmental and transcriptome analysis that Homo sapiens cryptochrome--1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. It is concluded that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism (Vieira, 2012).

Effects of Mutation or Deletion

A new rhythm mutation, cryb, has been isolated based on its elimination of period-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 proteins oscillate normally in temperature cycles). Consistent with the fact that light normally suppresses Tim protein, 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 (Stanewsky, 1998).

The cryb mutant does not exhibit phase shifts in response to light pulses. To assess clock resetting by brief pulses of light in (otherwise) constant darkness, phase response curves (PRC) were generated. Wild-type flies (and organisms in general) show phase delays after light pulses are given in the early subjective night, advances in late subjective night, and little or no phase shifting following pulses during the subjective day. When cryb flies are subjected to light pulses, no clear phase shifts result. This seems to contradict the fact that cryb flies tested for entrainment to different (phase shifted) LD cycles are able to "shift over" even at much lower light intensities. The apparent discrepancy could be explained by differences between the two experimental designs: in one case, flies are exposed to 12 hr of light, and in the PRC case, to only 10 min worth. In a very different kind of behavioral test involving responses to visual stimuli -- using short exposures of cryb flies to a relatively high light level, as in the PRC experiment -- the mutant exhibits normal optomotor behavior (Stanewsky, 1998).

The cry mutation does not eliminate cycling of Tim and Per protein levels within certain clock gene-expressing neurons. The cryb mutant exhibits rhythmic behavior in constant darkness in spite of the fact that no rhythmic protein expression during and after light entrainment is detectable. In Western blots involving head extracts, Per and Tim are measured mainly in photoreceptor cells (~90% of the anterior PNS and CNS cells expressing these genes. It was thought that rhythmic clock gene expression in the central pacemaker cells [the lateral neurons (LNs) that subserve behavioral rhythmicity (Kaneko, 1998)] could be masked by constitutive PER and TIM levels in the cryb mutant's eyes. The LNs consist of two groups of cells in each side of the brain, ~6 neurons in a relatively dorsal cluster (LNds) and ~10 such cells in a more ventrally located one (LNvs). Clock functions in the LNvs (along with the relevant molecular, physiological, and anatomical outputs) are probably sufficient to generate rhythmic behavior (Kaneko, 1998). Tim and Per expression were examined in the CNS (and in other cells of fly heads) by performing antibody stainings on sections of wild-type and cryb tissues. These were stained at two time points when Per and Tim each reached trough and peak levels. The staining intensities for different Per- and Tim-expressing cell types (compound-eye photoreceptors, glia, LNds, and LNvs) were scored blindly. Both proteins are observed to cycle in the LNs of cryb mutant flies, although with reduced amplitude as compared to wild type. Temporally constitutive, intermediate-level signals are observed in the eyes and glial cells, which explains the Western blot results obtained from extracts of cryb heads (Stanewsky, 1998).

The RNA oscillations observed for CRY mRNA (see cry Biological Overview) suggest that cry is a clock gene, and the primary sequence and light regulation indicate a role in photoreception. To link cry to circadian behavior, the GAL4 system was used to overexpress Cry in cells that govern locomotor activity rhythms. A newly generated tim promoter-GAL4 strain was crossed with a UAS-cry cDNA strain to generate progeny that should overexpress Cry in lateral neurons. Indeed, CRY mRNA levels are temporally constant and approximately 20-fold higher than the normal ZT1 peak level. The protein is also overexpressed (at least 30-fold at each time point) and cycles robustly, as expected from its light sensitivity. To quantitatively measure circadian light perception, flies were subjected to an anchored phase response curve (PRC) protocol; after entrainment to an LD cycle, flies were exposed to saturating or nonsaturating light pulses and the effect on behavioral phase measured. Control wild-type flies undergo a phase delay when the pulse is administrated during the early night and a phase advance during the late night. The overexpression had no significant effect on the period or strength of the locomotor activity rhythm, and there was no consistent or dramatic effect on the phase shift observed at high light intensities. However, the Cry overexpression strain is much more sensitive to light at low intensities. Especially in the delay zone, at ZT15, this effect is reproducibly very strong and suggests that Cry levels are normally limiting at low light intensities. In the advance zone, at ZT21, the magnitude of the effect is somewhat more variable from experiment to experiment. The striking light regulation of protein levels in clock-mutant as well as wild-type flies also indicates that Cry acts upstream of all known central pacemaker components (Emery, 1998).

Cryptochrome proteins are critical for circadian rhythms, but an understanding of their function(s) is uncertain. A mutation in Drosophila cryptochrome (dCRY) blocks an essential photoresponse of circadian rhythms, namely arrhythmicity under constant light conditions. This study concludes that dCRY acts as a key photoreceptor for circadian rhythms and that there is probably no other comparable photoreceptor in this species (Emery, 2000a).

Constant light causes the intrinsic circadian period of diurnal animals to shorten and that of nocturnal animals to lengthen (Aschoff's rule). More intense light produces more extreme effects, ultimately resulting in arrhythmicity in most mammals and birds. The circadian period of arthropods generally lengthens in constant light, whether the animal is nocturnal or diurnal. Drosophila melanogaster is no exception and intense constant illumination leads to arrhythmicity (Emery, 2000a).

The cryptochrome family includes blue-light photoreceptors. The single known Drosophila cryptochrome is thought to be a circadian photoreceptor: flies carrying a mutant allele, cryb, have severely decreased circadian photoresponses, whereas overproduction of dCRY causes increased photosensitivity. In mammals, however, mCRY1 and mCRY2 are more likely to be involved in the central clock mechanism (Emery, 2000a).

This raises the possibility that dCRY effects on photosensitivity reflect a role downstream of circadian photoreception, somewhere along the circadian light-input pathway or within the Drosophila central clock itself. This fits with the fact that cryb flies are still able to reset their circadian rhythm (entrain) to new light-dark cycles. cryb flies, however, remain behaviorally rhythmic in intense constant light, in contrast to wild-type flies and many other species that are arrhythmic under such conditions (Emery, 2000a).

The arrhythmicity of cryb flies must be a property of the cry gene, because the normal phenotype can be rescued by expressing wild-type dCRY in rhythm-generating cells of cryb flies. In intense constant light, the cryb mutant's behavior is strikingly similar to that of wild-type flies in constant darkness. An identical 24.7-hour period is also recorded under constant-darkness conditions, indicating that this slightly longer period is a characteristic of the background genotype. Thus, there is not even a detectable lengthening of period in the cryb mutant strain under constant light conditions. The free-running, circadian nature of cryb flies' constant-light behavior is further demonstrated by the 19.2-h period observed for flies with cryb in combination with a short allele of the period gene (per s) (Emery, 2000a).

These results show that the cryb mutation impairs the circadian photoreception pathway so profoundly that the fly cannot 'see' constant light. This mutant also responds very poorly to short light pulses; by these criteria, this circadian photoreceptor must be unique in Drosophila. How then can cryb flies entrain to different 24-h light-dark cycles? The missense cryb mutation might generate a protein with weak activity that would be sufficient for light-dark entrainment but not for a normal arrhythmic behavioral response to constant illumination. However, previous results suggest a different explanation: entrainment of cryb flies is through a second, completely separate light-input pathway. Visual photoreception may even directly influence locomotor activity, which then affects circadian rhythms only indirectly through a non-photic phase-resetting pathway (Emery, 2000a).

These results indicate that dCRY is an important circadian photoreceptor and probably the only dedicated one in Drosophila. Although additional central-clock functions for dCRY cannot yet be excluded, the abrogation of constant-light effects in cryb mutant flies indicates that this cryptochrome makes a unique contribution to Drosophila circadian photoreception (Emery, 2000a).

cryptochrome is an important clock gene. Recent data indicate that it encodes a critical circadian photoreceptor in Drosophila. A mutant allele, cryb, 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 cryb 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 pacemaker and a behavioral output system (Emery, 2000b).

The mutant cryb gene causes profound circadian light-response problems. While entrainment to 24 hr LD cycles still takes place in the mutant background, even this aspect of cryb circadian light perception is aberrant, because the mutant flies need much longer to entrain to a new cycle. A detailed examination of these LD entrainment data suggests that Cry contributes principally to adjusting the evening activity peak. The other major source of entrainment light information, the eyes, contributes principally to adjusting the morning peak. This fits with previous LD activity profile observations indicating that the phase of the evening activity peak is under clock control, whereas the phase of the morning peak is less sensitive to central clock mutations and is probably timed relative to some fixed environmental signal, e.g., the lights on or lights off transition. There is, however, a caveat, as the norpA; cryb double mutant phenotype suggests some interplay between the two light input pathways. The dual light input pathway for LD entrainment contrasts with the apparently unitary Cry input pathway for all other circadian photoresponses. This underscores the different nature of parametric (LD cycle) and nonparametric (short light pulse) entrainment. Taken together with the absence of any biological effect of CryB overexpression and the absence of CryB abundance cycling under LD conditions, cryb is probably a strong hypomorphic allele that encodes a protein without photoreceptor activity. This conclusion is consistent with the results of CryB expression studies in heterologous systems (Emery, 2000b and references therein).

Behavioral rescue experiments show that all known photoresponse defects of cryb are substantially rescued by expressing Cry only in the LNvs (ventral group of lateral neurons). The Cry-LNvs rescue is partial for the light pulse and constant light phenotypes, whereas it is almost complete for the LD entrainment defects of perS; cryb and norpAP41; cryb double mutant genotypes. In situ mRNA hybridization results suggest further that larvae also rely on Cry expression in the LNv precursor cells for circadian photoreception. There is no behavioral effect of Cry overexpression in the eyes, suggesting that the visual entrainment pathway is Cry independent (Emery, 2000b).

There are several possible explanations for the incomplete rescue of phase resetting and constant light arrhythmicity. pdf-GAL4 may be a relatively weak driver, such that Cry expression does not reach a required threshold level for full rescue. Cry may play a developmental role that is not fulfilled with pdf-GAL4-driven expression. There may also be other Cry-relevant cells, in addition to the LNvs, that contribute to circadian behavior. Consistent with this view, disco mutant flies lack LNvs but stay rhythmic for 1-3 days in DD. Cry expression studies should identify these accessory pacemaker cells. Good candidates are the dorsal lateral neurons and the dorsal neurons, both of which express Per and Tim and send projections to the same region of the brain as the LNvs (Emery, 2000b).

Why does Cry expression seem to be so high in LNvs, compared with other tissues, like the eyes? One possibility is that these neurons, located deep inside the brain, need to express high Cry levels to detect low light intensities. For example, this would allow the clock to respond at dawn and adjust its phase every day. Another explanation, more provocative perhaps, is that a high Cry concentration contributes to special pacemaker cell properties of the LNvs. In cryb, only the LNvs manifest Tim and Per cycling. This might reflect the high Cry levels in these cells, as well as a second, nonphotoreceptor contribution of Cry to pacemaker function. This Cry dark function could be to maintain the circadian oscillations of the molecular pacemaker, e.g., by contributing directly to the negative feedback loop, as shown in mammals. A true cry null mutation might therefore result in arrhythmicity, as observed in mammals (Emery, 2000b).

The evidence suggests that Cry contributes in a cell-autonomous manner to the Per/Tim molecular cycles. This presumably reflects independent photoreception of cells and tissues. In the periphery, Cry is absolutely required for light-dependent Tim degradation. Per, Tim, and Cry colocalization is not yet documented, but several studies have shown that cry is expressed in the body, as well as in different fly organs that contain autonomous clocks (Emery, 2000b).

Evidence is accumulating that the cell-autonomous property of circadian rhythms is universal, but with interesting differences between systems. The neuro-hormonal regulation of physiology may limit the autonomy of peripheral oscillators in mammals, where the suprachiasmatic nucleus (SCN) appears to be the principal central mammalian pacemaker organ. But the SCN, as well as most internal clocks and tissues, is probably not directly light sensitive. It receives photic cues from the eyes, where the circadian photoreceptor molecule has not been identified. Moreover, it is unclear whether mammalian Crys ever function as cell-autonomous photoreceptors, e.g., in cultured retina cells that exhibit circadian oscillations (Emery, 2000b).

Mammalian peripheral oscillators are probably under SCN control, largely through humoral connectors. This scheme accounts for the 4 hr phase difference between peripheral and SCN molecular cycles in vivo. The lack of any reported phase difference in Drosophila, i.e., between the molecular cycles in the periphery and LNvs, is consistent with rescue experiments and presumably reflects independent cell-autonomous connections by Cry to environmental light cues. Remarkably, this includes even the LNvs, since Cry expression within these brain pacemaker cells controls every known aspect of circadian behavioral photosensitivity. Although there is evidence in other systems for deep brain photoreceptors, Cry is the only functionally identified light sensor of this kind. Taken together with the recent identification of a behavioral output factor within the LNvs, they are now known to contain all three components of a functional, cell-autonomous circadian clock: photoreception, a central pacemaker, and well-defined output (Emery, 2000b).

It is concluded that all three clock components are present in the Drosophila circadian pacemaker cells. To control behavioral locomotor activity in a circadian manner, three elements are required: an input pathway, a pacemaker, and an output pathway. All three are present in the LNvs. By expressing Cry, the LNvs are directly sensitive to light. Cry controls the phase of the pacemaker, which is composed of a transcriptional feedback loop involving Per/Tim and Clock/Cyc dimers. The former regulates the transactivation potential of the latter in the nucleus. Doubletime, a kinase, is also necessary for central pacemaker function. This transcriptional loop regulates, through poorly understood mechanisms that may involve Vrille, the expression and the release of the neuropeptide PDF, which is an important output element of this circadian system (Emery, 2000b).

Cryptochromes are flavin/pterin-containing proteins that are involved in circadian clock function in Drosophila and mice. In mice, the cryptochromes Cry1 and Cry2 are integral components of the circadian oscillator within the brain and contribute to circadian photoreception in the retina. In Drosophila, cryptochrome (CRY) acts as a photoreceptor that mediates light input to circadian oscillators in both brain and peripheral tissue. A Drosophila cry mutant, cryb, leaves circadian oscillator function intact in central circadian pacemaker neurons but renders peripheral circadian oscillators largely arrhythmic. Although this arrhythmicity could be caused by a loss of light entrainment, it is also consistent with a role for Cry in the oscillator. A peripheral oscillator drives circadian olfactory responses in Drosophila antennae. Cry contributes to oscillator function and physiological output rhythms in the antenna during and after entrainment to light-dark cycles and after photic input is eliminated by entraining flies to temperature cycles. These results demonstrate a photoreceptor-independent role for Cry in the periphery and imply fundamental differences between central and peripheral oscillator mechanisms in Drosophila (Krishnan, 2001).

Circadian rhythms can be entrained by light to follow the daily solar cycle. In adult flies a pair of extraretinal eyelets expressing immunoreactivity to Rhodopsin 6 each contains four photoreceptors located beneath the posterior margin of the compound eye. Their axons project to the region of the pacemaker center in the brain with a trajectory resembling that of Bolwig's organ, the visual organ of the larva. A lacZ reporter line driven by an upstream fragment of the developmental gap gene Kruppel is a specific enhancer element for Bolwig's organ. Expression of immunoreactivity to the product of lacZ in Bolwig's organ persists through pupal metamorphosis and survives in the adult eyelet. It is thus demonstrated that the adult eyelet derives from the 12 photoreceptors of Bolwig's organ, which entrain circadian rhythmicity in the larva. Double labeling with anti-pigment-dispersing hormone shows that the terminals of Bolwig's nerve differentiate during metamorphosis in close temporal and spatial relationship to the ventral lateral neurons (LNv), which are essential to express circadian rhythmicity in the adult. Bolwig's organ also expresses immunoreactivity to Rhodopsin 6, which thus continues to be expressed in the adult eyelet. Action spectra of entrainment were compared in different fly strains: in flies lacking compound eyes but retaining the adult eyelet (so1), lacking both compound eyes and the adult eyelet (so1;gl60j), and retaining the adult eyelet but lacking compound eyes as well as Cryptochrome (so1;cryb). Responses to phase shifts suggest that, in the absence of compound eyes, the eyelet together with Cryptochrome mainly mediates phase delays. Thus a functional role in circadian entrainment first found in Bolwig's organ in the larva is retained in the eyelet, the adult remnant of Bolwig's organ, even in the face of metamorphic restructuring (Helfrich-Forster, 2002).

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. 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 glass60j;cryb double mutants, which lack all known external and internal eye structures in addition to being devoid of cryptochrome (Helfrich-Forster, 2001).

Extraocular photoreception is involved in the circadian systems of many organisms, but in most of them the structures and molecules involved are unknown or barely characterized. The present study demonstrates that nonretinal photoreceptors are involved in entrainment of Drosophila's circadian clock: fruit flies utilize at least a tripartite light-input pathway -- one pathway makes use of cryptochrome; a second acts through norpA-dependent photoreceptors in the compound eyes (perhaps also the ocelli), and a third pathway, which acts independently of norpA and cry gene functions, seems likely to involve the extraretinal Hofbauer-Buchner eyelets or other extraocular photoreceptors located in the brain (Helfrich-Forster, 2001).

These separate clock-input pathways influence the circadian system in different ways. Light input through CRY mainly entrains the evening peak of behavioral activity. Retinal and extraretinal eye structures predominantly synchronize the morning peak of activity. In spite of their different effects on behavioral rhythmicity, each light-input pathway alone seems capable of entraining the locomotor rhythm in a rather normal manner when the other one is impaired. Only when all three input routes -- those subserved by CRY, the compound eyes/ ocelli, and extraretinal eye structures are absent is the circadian system of the fly unable to respond to light. This is so at the cellular and at the behavioral level, thus revealing that fruit flies entrainment to multiple light-input pathways for adapting their circadian clock to the cyclic environmental LD changes. Interestingly, similar findings have recently been described for mice, for which depletion of retinal photoreceptors results in almost complete circadian blindness (Helfrich-Forster, 2001).

How Drosophila's multiple photoreceptors might interact remains a mystery but is likely related to the task, faced by many organisms, of extracting time-of-day information from dawn and dusk. During natural twilight, the quality of light changes in three important respects: the amount of light, its spectral composition, and the direction of incoming light (i.e., the position of the sun). These photic parameters all change in a systematic way at twilight times ['Heavenly shades of night are falling, it’s twilight time' (The Platters, 1958)]; all could be used by the circadian system throughout times of changing photic conditions at dawn and dusk, thus forming a versatile input system that subserves daily adjustments of the rhythm’s pace. Using different rhodopsins in addition to CRY permits the fly systems to scan all the way from the UV into the red (Helfrich-Forster, 2001).

The observation that the different pathways have at least some overlapping cellular targets provides a first hint about how the fly's different photoreceptors communicate: PER cycling in the s-LNv brain cells can be at the very least a tripartite light-input pathway -- one of them makes use of cryptochrome; a second pathway acts through internal eye structures (in the glass mutant). Similarly, in the absence of CRY and functional external eyes (in the norpAP41;cryb double mutant), the same neurons are acts independently of norpA and cry gene functions, synchronized by extraretinal photoreceptors. Such multiple input aimed at a single cell type would allow the organism to integrate the incoming light (Helfrich-Forster, 2001).

Among the multiple photoreceptors that could contribute to circadian photoreception in Drosophila, the contributions of the external eyes and of CRY function have been demonstrated with the help of the norpAP41;cryb doubly mutant flies that show entrainment defects, which are much more severe than those exhibited by either mutant alone. Nevertheless, this double-mutant type is not completely blind in the circadian sense, compared with the effects of the gl60j;cryb combination revealed in this study. Most norpAP41;cryb individuals are still able to entrain to LD cycles (Helfrich-Forster, 2001).

There are at least two possible explanations for these findings: cryb is not a loss-of-function mutation (indeed, this is a missense mutant), or other norpA-independent photoreceptors feed into the clock. The fact that flies could be generated that are totally circadian blind favors the second hypothesis (Helfrich-Forster, 2001).

The H-B eyelets send their projections directly to the accessory medulla and are therefore anatomically well suited to transmit light signals to the LNv pacemaker cells. The fact that, in the absence of CRY, PER cycling in the s-LN cells is nicely entrained also favors this hypothesis. The H-B eyelets express the photopigment Rhodopsin 6 and thus seem to have photoreceptive properties. The input pathway via Rhodopsin 6 might utilize the fly’s norpA-independent phospholipase C (which is expressed in many neurons in its phototransduction cascade (Helfrich-Forster, 2001).

Further candidates for circadian photoreceptors (revealed in this study) are the clock-gene-expressing dorsal neurons called DN1 cells. Like the photoreceptor cells of the compound eyes, the ocelli and the H-B eyelets, the DN1s appear to be eliminated by the gl60j mutation. Similar to the H-B eyelets, the DN1s send axonal projections toward the LNvs and could entrain the latter through this anatomical pathway. Furthermore, disconnected mutant flies, which largely lack the LNv but not the DN1 cells, are able to entrain to LD cycles. However, it is not known whether the DN1s express a photopigment or have photoreceptive properties like those exhibited by the H-B eyelets (Helfrich-Forster, 2001).

In spite of the inability of the gl60j;cryb double mutant to entrain to LD cycles, the behavior of such flies was still modified by the altered environmental conditions. This modulation of the activity level is interpreted as direct effects of light/radiant energy on locomotion that bypass the circadian system. Light-related energy often exerts such direct (or masking) effects on physiological parameters, including behavior. There are possibilities to distinguish real entrainment from masking: (1) after a phase shift of the LD cycle, the circadian rhythm often takes several transient cycles to reentrain to the new LD regime, whereas masking follows the new light schedule immediately (in Drosophila, transients can be observed at very low light intensities or in photoreceptor mutants like cryb; (2) after transfer into constant darkness, masking disappears immediately whereas an entrained rhythm starts to free run from the phase it had established in LD, and (3) masking is independent of a functional circadian clock -- for example, it occurs in animals deprived of their clock, such as squirrel monkeys suffering from lesions of their suprachiasmatic nucleus arrhythmic per0 mutants of Drosophila (Helfrich-Forster, 2001).

In gl60j;cryb, prominent masking is observed at the highest light intensities employed (1000 lux). The sudden increase of the activity level after lights off and phase delay of the LD cycle did not show any transients after the 8 hr phase shift. Furthermore, this apparently forced activity disappears immediately after transfer into DD and independent of a functional clock, owing to its presence in these doubly mutant flies, which exhibit apparent rhythmicity in constant darkness. Moreover, no entrained cycling of clock protein levels was observed in these gl60j;cryb flies, demonstrating that the forced behavior is neither the consequence of molecular clock gene cyclings nor a nonphotic Zeitgeber that could influence the circadian clock (Helfrich-Forster, 2001).

In summary, the circadian blindness of flies expressing both glass and cryptochrome mutations is due to elimination of all photoreceptor cells that participate in entraining the circadian system. Similar complex light entrainment pathways may also exist in vertebrates. Interestingly, cryptochromes, certain opsins located in the retina, and standard photoreceptor cells are candidates for participating in the circadian photoreception of mammals. Thus, rather than having an exclusive photopigment for entrainment of circadian rhythms, the situation in mammals could be similar to that in Drosophila: multiple photoreceptors share the workload involved in transmitting the principle environmental Zeitgeber to the circadian clock (Helfrich-Forster, 2001).

Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait

Identifying the genes involved in polygenic traits has been difficult. In the 1950s and 1960s, laboratory selection experiments for extreme geotaxic behavior in fruit flies established for the first time that a complex behavioral trait has a genetic basis. But the specific genes responsible for the behavior have never been identified using this classical model. To identify the individual genes involved in geotaxic response, cDNA microarrays were used to identify candidate genes and fly lines mutant in these genes were assessed for behavioral confirmation. The identities of several genes that contribute to the complex, polygenic behavior of geotaxis have thus been determined (Toma, 2002).

Pioneering experiments on Drosophila melanogaster and Drosophila pseudoobscura investigated the nature of the genetic basis for extreme, selected geotaxic behavior. These experiments constituted the first attempt at the genetic analysis of a behavior. Selection and chromosomal substitution experiments successfully showed that there is a genetic basis for extreme geotaxic response in flies and, by implication, for behavior in general. These experiments also added to understanding of the role of variation in phenotypic evolution and selection. Despite their seminal contributions in behavioral genetics, population genetics and the study of selection, by their nature these experiments could not identify specific genes (Toma, 2002 and references therein).

These results highlight both the success and the limitation of behavioral selection experiments. Although selection results tend to be representative of the natural interactions of genes that produce behavior and can demonstrate that a trait has a genetic basis, they do not pinpoint specific genes that influence the trait. This is partly due to the involvement of many genes and the relatively minor role of each in complex polygenic phenotypes -- a problem that is especially acute for the intrinsically more variable phenotypes that are associated with behavior. The advent of cDNA microarray technology offers an easily generalized strategy for detecting gene expression differences and can complement other means of identifying the genes that underlie complex traits. An expression difference may occur in a gene that is not itself polymorphic, but that gene may contribute to the realization of the phenotypic difference (Toma, 2002).

As a starting point for identifying genes that affect a complex trait, the selected, established Hi5 and Lo extreme geotaxic lines were examined for changes in gene expression between strains of Drosophila melanogaster subjected to long-term selection and isolation. A two-step approach was used: (1) the differential expression levels of mRNAs isolated from the heads of Hi5 and Lo flies was determined using cDNA microarrays and real-time quantitative PCR (qPCR); (2) a subset of the differentially expressed genes was independently tested for their influence on geotaxis behavior by running mutants for these genes through a geotaxis maze. It was reasoned that some of the differences in gene expression between strains might be related to phenotypic differences and that it should therefore be possible, at least in part, to reconstruct the phenotype with independently derived mutations in some of the differentially expressed genes (Toma, 2002).

The findings indicate that differences in gene expression can be used to identify phenotypically relevant genes, even when no large, single-gene effects are detectable by classical, quantitative genetic analysis. Three of the four genes implicated by microarray and qPCR measurements caused differences in geotaxis, whereas none of the six control genes had an effect. Only those genes that had larger differences in expression according to the microarrays, or that were significantly different according to qPCR results (cry, Pdf and Pen), significantly changed geotaxis scores. The converse was not true, because altered geotaxis behavior did not always accompany larger differences in mRNA levels, as shown by pros, although this might reflect the sensitivity of pros to aspects of the genetic context. All of the genes tested for which there was little or no difference in mRNA levels between the selected Hi5 and Lo lines also showed no influence on geotaxis behavior (Toma, 2002).

The directionality of behavioral and mRNA differences proved to be consistent with predictions that were based on expression levels. Homozygous null mutants of Pdf and cry showed a significant increase in geotaxis score, which is consistent with a lower level of expression of these genes in Hi5 relative to Lo. Similarly, the heterozygous Pen mutant showed a significant downward shift in geotaxis score, which is consistent with a lower level of Pen expression in Lo relative to Hi5. Thus, the change in behavior of the tested mutants corresponds to the direction predicted by differences in transcript level in the selected Hi5 and Lo lines (Toma, 2002).

Whereas the cry, Pen and female Pdf mutants produced the anticipated effect on behavior, the magnitude of behavioral effect was smaller than in the original selected lines. This probably reflects the difference between the aggregate effect of an ensemble of genes in the selected lines as opposed to the individual effect of a single mutant gene in a neutral background. In addition, their relatively small effects are exactly the results that one would predict in a polygenic system such as geotaxis behavior, in which many genes have small contributions to the overall phenotype. The three genes identified in this study would not have been predicted on the basis of their previously defined functions (Toma, 2002).

These results show that the two separate approaches to behavioral genetics -- the classical Hirschian quantitative analysis of genetic architecture and the modern Benzerian approach of single-gene mutant analysis -- are complementary and can be unified. This study used the results of a Hirschian approach of laboratory selection for natural variants to identify single gene differences, such as one would find in a Benzerian approach. The results are consistent with the suggestion that naturally occurring variants in behavior correspond to mild lesions in pleiotropic genes (Toma, 2002).

Finally, the results show that differences in gene expression identified by cDNA microarray analysis can be used as a starting point for narrowing down the numbers of candidate genes involved in complex genetic processes. Such an approach is analogous, as well as complementary, to the current method of mapping quantitative trait loci to large chromosomal intervals and then making educated guesses about which genes within those intervals may be involved in the trait (Toma, 2002).

The combination of selection, with its ability to exaggerate natural phenotypic variation, and global analysis of differences in gene expression by cDNA microarray analysis offers a promising approach to previously intractable molecular analyses of behavior. The geotaxis genes that were identified might have been the direct targets of selection, or they might be downstream of the direct targets. Additional studies using the Hi5 and Lo selected lines will be required to distinguish between these possibilities and to determine the causal role that these genes have in the context of the selected lines (Toma, 2002).

This study has gone from the selection of a 'laboratory-evolved' behavioral phenotype, to screening for mRNA differences, to partially reconstituting the phenotype using mutants. This shows the feasibility of combining genomic and classical genetic approaches for the breakdown and partial reassembly of an artificially selected behavioral trait (Toma, 2002).

Effects of combining a cryptochrome mutation with other visual-system variants on entrainment of locomotor and adult-emergence rhythms in Drosophila

Photoreception is an important component of rhythm systems and is involved in adjusting circadian clocks to photic features of daily cycles. In Drosophila, it has been suggested that there are three light input pathways to the clock that underlie rhythms of adult behavior: One involves the eyes; the other two involve extraocular photoreception through a structure called the Hofbauer-Buchner (H-B) eyelet and light reception carried out by pacemaker neurons themselves, mediated by a substance called cryptochrome. All photoreceptor cells including the H-B eyelet have been surmised to be removed by glass-null mutations. Mutations in the no-receptor-potential-A (norpA) gene cause the compound eyes and ocelli to be non-functional and may also affect the eyelet's function. The one cryptochrome mutant known (cryb) harbors an amino-acid substitution in the blue-light absorbing protein encoded by this gene. With regard to adult locomotor rhythms, all single mutants (gl60j, norpAP41, and cryb) re-entrained to altered light:dark (LD) cycles in which the L phase involves relatively intense light. Dropping light levels ca. 10 or ca. 30-fold permits small percentages of doubly-mutant gl60j;cryb flies clearly to re-synchronize their behavior. The marginal re-entrainability in the lowest-light situation nevertheless involves superior responsiveness of the gl60j;cryb type, compared with that observed using a different re-entrainment protocol. Furthermore, transgenic types in which rhodopsin-expressing cells within the H-B eyelet are ablated or suffer from the effects of tetanus-toxin also entrain with behavior similar or superior to that of gl60j;cryb at a low light level. Light inputs that are necessary to synchronize periodic adult emergence can be inferred to involve a cry-dependent pathway and perhaps also a norpA-dependent one, so that combining mutations in these two genes would cause cultures to be unentrainable. The current results show that each singly-mutant type ecloses rhythmically; flies emerging from norpAP41;cryb cultures also (on balance) exhibit solid eclosion rhythmicity. The ensemble of these behavioral and adult-emergence results suggest that additional light-to-clock pathways function within the system; alternatively, that rhythm assays employed in this study have teased out residual function of the mutated Cru protein (Mealey-Ferrara, 2003).

Seasonal behavior in Drosophila melanogaster requires the photoreceptors, the circadian clock, and phospholipase C

Drosophila locomotor activity responds to different seasonal conditions by thermosensitive regulation of splicing of a 3' intron in the period mRNA transcript. The control of locomotor patterns by this mechanism is primarily light-dependent at low temperatures. At warmer temperatures, when it is vitally important for the fly to avoid midday desiccation, more stringent regulation of splicing is observed, requiring the light input received through the visual system during the day and the circadian clock at night. During the course of this study, it was observed that a mutation in the no-receptor-potential-A(P41) (norpA(P41)) gene, which encodes phospholipase-C, generates an extremely high level of 3' splicing. This cannot be explained simply by the mutation's effect on the visual pathway and suggests that norpA(P41) is directly involved in thermosensitivity (Collins, 2004).

The proportion of per transcripts that were spliced at 18°C and 29°C, averaged over several LD 12:12 cycles was examined in Canton-S WT and per01, tim01, cryb, and per01; cryb mutant backgrounds. In all backgrounds splicing levels fall as the temperature rises, with 40%-60% of transcripts spliced at 18°C and 20%-45% at 29°C. However, not all genotypes react in the same way to temperature changes (Collins, 2004).

The smallest but nevertheless significant effect of temperature on splicing levels is observed in per01; cryb, suggesting that the temperature-sensing system for splicing may be compromised in the double mutant. A significant temperature x time effect reveals that the temporal patterns of cycling differ among temperatures, and the absence of any other significant interactions suggests that all genotypes respond similarly. There is very little evidence for a significant day/night cycle in the proportion of per transcripts that are spliced at 18°C, but at 29°C, all genotypes reveal a higher level of splicing post lights off (ZT12) compared to the trough at ZT8. At 18°C, the per01 and tim01 mutations have no significant effect on the level of splicing of per mRNA compared to WT. However in cryb flies, splicing levels are significantly elevated, particularly after lights off. This is also the case when per01; cryb is compared to WT. At 29°C, splicing levels are generally 5%-10% higher in per01, tim01, and cryb mutants compared to WT in the light, but 15%-20% higher after lights off at ZT12. This suggests that in the presence of light, splicing levels are reduced due largely to a clock-independent mechanism. In darkness, the clock and Cry become critical for maintaining this low splicing level at high temperatures (Collins, 2004).

The double mutant per01; cryb shows a highly significant increase in splicing of ~20% throughout the day/night cycle compared to WT. Thus, at high temperature, either the presence of the circadian photoreceptor Cry or a functional circadian clock is sufficient to largely repress daytime splicing. With both eliminated, daytime splicing levels are elevated. In contrast, repression of splicing in the absence of light requires the circadian clock plus Cry. It seems somewhat counterintuitive that Cry, which is activated by light, plays a more prominent role in repressing splicing at night than it does during the day (Collins, 2004).

Cry is likely to be a dedicated circadian photoreceptor yet at 29°C, splicing is repressed during the light phase even in cryb. This suggests that the light input to the splicing machinery cannot be primarily mediated by Cry. To confirm that light represses splicing, the effects that short photoperiods and constant darkness (DD) have on splicing levels in WT was investigated. There is a significant effect of reducing photoperiod on the splicing level with an elevated level of splicing in DD compared to LD 12:12, and similarly in LD 6:18, splicing levels are enhanced. Because the repression of splicing by light in LD 12:12 at 29°C does not require the presence of Cry, whether the visual system plays a role in setting the splicing level was investigated by examining the splicing of per transcripts in the mutants gl60j and norpAP41 (Collins, 2004).

The proportion of per mRNA transcripts that are spliced at both temperatures is increased in both the norpAP41 and gl60j backgrounds compared to WT. At 18°C, ~65% of per transcripts are spliced in norpAP41 and ~60% in gl60j, whereas at 29°C, these levels fall to ~55% and ~40%, respectively. Apart from a marginal difference between norpAP41; cryb, and norpAP41 at 18°C, there are no significant effects for either norpAP41 or gl60j when combined with cryb. These results indicate that the visual system rather than Cry is primarily responsible for the light-dependent repression of splicing. Unlike WT, per splicing levels do not rise after lights off at 29°C in either norpAP41 or gl60j (Collins, 2004).

Interestingly, in gl60j, there is a 20% difference between the splicing levels at different temperatures (60%-40%), whereas in norpAP41, this difference is reduced to 10% (65%-55%). The difference in gl60j is similar to that seen in WT (45%-25%). Thus per splicing in norpAP41 is relatively insensitive to temperature changes. It is also clear that the level of splicing in norpAP41 is significantly higher at all times and temperatures than gl60j. Therefore, the effect of norpAP41 on splicing is greater than that of gl60j, despite gl60j being the more severe visual mutant (Collins, 2004).

Locomotor activity profiles of all genotypes were also monitored at 18°C and 29°C. Because each genotype shows a higher level of splicing at 18°C than at 29°C, it would be predicted that this would generate an earlier evening activity peak at 18°C. This is the case for WT, cryb and norpAP41, but not for norpAP41; cryb or gl60j, where despite elevated splicing levels at higher temperatures, there is no difference in the phase of activity. gl60j cryb does not entrain to LD cycles at 25°C, so was not included in this analysis (Collins, 2004).

The average proportion of per transcripts that are spliced at 18°C rises from WT (45%) to cryb (50%) to gl60j (60%) to norpAP41 (65%), and at 29°C from WT (25%) to gl60j and cryb (~35%) to norpAP41 (55%). If the per splicing level is the only determinant of evening locomotor peak position, then a similar progression in the timing of this peak would be expected. The evening activity peaks of these different genotypes at 18°C and 29°C were compared. For norpAP41, cryb, and WT, there is an inverse relationship between average splicing levels and the position of the activity peak at 18°C, with norpAP41 and cryb having similarly advanced activity peaks compared to WT. At 29°C, the same inverse relationship holds, with norpAP41 advanced compared to cryb, which is in turn earlier than WT. Thus, those genotypes that show temperature-dependent changes in their evening activity generally display a correlation between average per splicing levels and the timing of the evening activity peak of the following day. Conversely, norpAP41; cryb and gl60j,, which show no significant differences in the phase of evening activity at different temperatures, have high splicing levels but relatively delayed evening activity peaks (Collins, 2004).

These observations raise the question of why the splicing level does not always relate to the timing of the evening locomotor activity peak, as in gl60j and norpAP41; cryb. Thus the per RNA profiles of gl60j and norpAP41 were compared to WT. Because per does not cycle in cryb whole head homogenates, the underlying cycle in this background was not examined. WT and norpAP41 show similar profiles, with an earlier per mRNA peak and higher overall level of per at the lower temperature. In contrast, there is no cycle in gl60j at either temperature, and levels of per are significantly different from WT and norpAP41 (Collins, 2004).

Therefore, to entrain locomotor behavior to different seasons, the fly's clock must respond to changes in both light and temperature. This is mediated through a molecular switch, whereby increases in temperature repress the splicing of an intron within the 3' UTR of per, delaying the onset of evening locomotor activity. Light also represses splicing, with higher splicing levels seen in shorter photoperiods, allowing locomotor activity to be fine-tuned to any given set of photoperiodic and temperature conditions. During the first day of DD, the level of splicing rises continuously. This is presumably because at the beginning of DD, the level of splicing is set low from the previous day's light input. Normally the light from the next day maintains this repressed level of splicing, but because this light input is absent, the repression of splicing is lifted, leading to a gradual rise in splicing levels (Collins, 2004).

The most obvious source for light input into the splicing machinery is the circadian photoreceptor Cry. However, analysis of the splicing levels in cryb shows that, although this mutation has an effect on splicing levels at 18°C, this effect is marginal and is seen only after lights off. This implies (1) that any function of Cry in the repression of splicing is not via the activation of this molecule by light; (2) because Cry is relatively dispensable for circadian locomotor rhythmicity per se, it also suggests that any minor role in splicing at low temperature is unrelated to the functioning of the clock. As the temperature rises, Per, Tim, and Cry all become involved in the regulation of per mRNA splicing. At 29°C, all three mutants show the same splicing phenotype, with ~30% of transcripts spliced during the day, but at night splicing is enhanced to ~45%. Although Per, Tim, and Cry are known to associate in light conditions, Cry and Tim can also associate in darkness, so it is not unexpected that the elimination of any one of the three proteins has a similar effect. Night time is also when the levels of these proteins are at their highest, and therefore any effects would be maximal (Collins, 2004).

At 29°C and in the presence of light, the levels of splicing in per01; cryb are elevated above those of either single mutant, which are themselves similar to WT. This suggests that the presence of either Per or Cry is required for light to repress splicing at 29°C. After lights off, the elevated levels of splicing of per are very similar in per01, tim01, cryb, and per01; cryb. Therefore Per, Tim, and Cry probably work together to repress splicing in the dark at 29°C. An alternative view for the virtually identical per01, tim01, and cryb splicing levels at 29°C is that this reflects a masking effect of light, so that exogenous LD cycles have a greater effect on splicing at night compared to WT, which shows a modest but significant day-night rhythm. Such stronger masking effects on locomotor behavior have also been observed in cryb mutants, but any mechanism that might relate or explain these observations remains obscure (Collins, 2004).

The examination of whole head homogenates means that the majority of biological material is derived from the eyes so may not represent exactly what occurs in the pacemaker neurons. The eyes are peripheral clocks, and the cryb mutation stops the cycling of the clock in whole head homogenates, although cycling continues in the pacemaker cells. One possibility is that the splicing observed in cryb does not truly reflect the role of Cry in setting splicing levels but is instead a consequence of the clock having stopped in the eyes, thus explaining why per01, tim01, and cryb all show the same splicing phenotype. However, if this splicing phenotype is simply what happens when the clock stops, then per01; cryb should show the same splicing phenotype as either single mutant. This is not the case, because the daytime splicing in per01; cryb at 29°C is dramatically elevated compared to either single mutant. Thus the splicing phenotypes of per01, tim01, and cryb cannot simply be a result of the clock having stopped. This means that it is the presence of these proteins, rather than their clock-dependent cycling, that is important to the regulation of per splicing levels (Collins, 2004).

In gl60j, there is no per mRNA cycle in whole head homogenates. This means that in the majority of cells in the gl60j head, the clock has either stopped or cells have become desynchronized. If the former is true, then splicing levels of gl60j should resemble those of per01 or tim01, and this is clearly not the case. If the latter is true, this could prevent the observation of any splicing rhythm, but the level of splicing observed should still represent the average level of splicing in this mutant background, which is clearly significantly different from WT. In any case, splicing levels observed in all visual mutants are likely to represent the effect of removing visual photoreception, because these elevated levels are similar to those observed in WT in DD (Collins, 2004).

norpAP41 and gl60j have considerably higher splicing levels than WT and cryb mutants at both temperatures, indicating that information received via the visual system rather than Cry drives this repression of splicing, which is borne out by analysis of gl60j cryb and norpAP41; cryb double mutants. The splicing levels of gl60j and gl60j cryb are similar at both temperatures, which is also true of norpA and norpAP41; cryb at 29°C. At 18°C, there is slightly more spliced per RNA in norpAP41; cryb than in the norpAP41 single mutant, reflecting the earlier result where cryb showed a marginal enhancement of splicing at cooler temperatures. These results also demonstrate that unspecific genetic background effects are not responsible for this marginal effect of cryb, because the double mutant background should make any interacting loci heterozygous. This lack of significant background effects in determining overall splicing levels has been confirmed by examining several natural European D. melanogaster lines. All mutants studied here show the same significantly enhanced splicing patterns when compared to any of the wild-caught isolates (Collins, 2004).

Unlike the clock and cryb mutants, there is no day-night difference in splicing levels at 29°C in either gl60j or norpAP41. One possibility is that visual system structures are required for the repression of splicing even in the dark, hence the overall elevated splicing levels in norpAP41 and gl60j at all times. This would be surprising, because such a role would obviously have to be light independent. More likely, the light input received through the eyes sets the splicing level during the day, and the clock maintains this repression at night. Thus, if the visual input is removed or reduced, as in DD, gl60j, or norpAP41 mutants, or in shorter photoperiods, then the subsequent splicing level is set higher. The difference in roles between cry and the visual system on per splicing levels may also partly explain recent observations that cryb mutants are able to adapt the timing of locomotor activity to long and short photoperiods, whereas flies with defective visual photoreception, including gl60j, are not (Collins, 2004).

Interestingly, although gl60j is the more severe visual mutant, norpAP41 has significantly higher per splicing levels than gl60j at both 18°C and 29°C. Additionally, whereas the difference between splicing levels at 18°C and 29°C is maintained in gl mutants (~65% and ~45% of transcripts spliced vs. ~45% and 25% in WT at 18°C and 29°C, respectively), this is greatly reduced in norpAP41 (65% and 55%). One possible explanation for this is that norpA may be a signaling molecule in the temperature-sensing pathway for the clock. The patterns of locomotor activity support a role for norpA in temperature sensing, with the norpAP41 fly's locomotor patterns seemingly more sensitive to high temperatures than WT. Additionally, norpAP41 evening locomotor activity peaks early at both 18°C and 29°C, and per mRNA splicing shows a corresponding elevation compared to WT. These are responses associated with low temperatures in WT D. melanogaster, and therefore norpAP41 mutants behave as if they have an impaired ability to detect high temperatures. norpAP41 flies still detect temperature changes (witness the altered evening peaks and splicing levels); they just react as if the temperature is colder than it actually is (Collins, 2004).

Thus, the enhanced per splicing seen in norpAP41 may reflect a direct link between norpA-encoded PLC signaling and the temperature sensitivity of the splicing mechanism, independent of norpA visual function. In the phototransduction cascade, rhodopsin activates a G-protein isoform that in turn activates the PLC encoded by norpA. As a result of this activation, Ca2+ permeable light-sensitive channels are opened, including members of the transient receptor potential (TRP) class. Recently it has been demonstrated that dANKTM1, a D. melanogaster TRP channel, is activated by temperatures from 24°C to 29°C. In addition, D. melanogaster painless mutant larvae have a disrupted TRP channel and display defective responses to thermal stimuli. Because several TRP family members act as thermal sensors in mammals, TRP channels appear to have an ancient heat-sensing function that is retained in both vertebrates and invertebrates. Given that this study has identified a heat-sensing role for norpA, and norpA is known to activate TRP channels in photoreception, it is not unreasonable to suppose that norpA plays a general role in responses to temperature stimuli (Collins, 2004).

per splicing levels may also impact on aspects of behavior other than the timing of evening locomotor activity. For instance, the free-running period of norpAP41 is ~1 h shorter than WT. The splicing levels of per mRNA are greatly elevated in this background, and elevated splicing is predicted to advance the Per protein cycle and thus speed up the clock. In fact, the splicing mechanism should have the effect of speeding up the clock at colder temperatures and slowing it down at high temperatures, thereby providing a potential basis for temperature compensation (Collins, 2004).

The position of the evening activity peak at different temperatures moves in different mutant backgrounds. For WT, norpAP41, and cryb, the level of splicing appears to correlate with the position of the evening activity peak at different temperatures. At 18°C, there is a small but significantly greater relative amount of spliced per RNA in cryb than in WT, resulting in the earlier evening activity peak seen in cryb flies. This difference in per splicing is greatest after lights off at both temperatures. This is when Per levels will be rising, because Tim is present for Per stabilization, so enhancement of Per accumulation by elevated per splicing is likely to have its most noticeable effect around dusk or early evening. A similarly consistent situation is seen in norpAP41: there is more spliced per mRNA present at 18°C (65%) than 29°C (55%), accounting for the earlier peak of evening activity at 18°C. Additionally these levels are higher than those seen in either WT (45% and 25% per transcripts spliced at each temperature) or cryb (55% and 40%) and relates to the earlier phases of locomotor activity seen in norpAP41 compared to the other genotypes. However, at 18°C there is more spliced per in norpAP41 than in cryb, but the evening activity peak occurs at the same time. The simplest explanation is that there is a limit to how early the evening activity peak can occur, no matter what the per splicing level, because splicing alters the accumulation of Per protein; this is limited by the light-dependent degradation of Tim. Therefore, in general, the level of splicing determines when the peak level of locomotor activity will occur (Collins, 2004).

The level of splicing of the per intron cannot be the only determinant of evening peak position, because the relationship between the per splicing level and evening activity peak position breaks down in norpAP41; cryb and gl60j, where there are different levels of splicing at the two temperatures but no corresponding difference in the evening peak position. When the underlying per mRNA cycles of gl60j, norpAP41, and WT flies were analyzed at 18°C and 29°C, it was found that whereas per levels cycle in norpAp41 and WT, this cycle is lost in gl60j. If there is no underlying per RNA cycle, then there is no mRNA peak to be advanced or delayed by splicing (Collins, 2004).

At the cellular level, although gl is not a clock component, when mutated, it eliminates a number of clock-expressing cells within the head, including the eyes, ocelli, Hofbauer-Buchner (H-B) eyelet, and the dorsal neuron 1 (DN1) cells. Despite this, the primary effect on the clock is to remove most of the visual entrainment pathway, but the clock in the key pacemaker cells of gl60j mutants must still be functional, because behavior still entrains to LD cycles and remains rhythmic in DD. It is significant that the crosstalk between different classes of clock cells is essential for the generation of robust behavioral rhythms. Thus loss of the overall per mRNA rhythm may be a consequence of disrupting this network in gl60j, and, while leaving the basic system intact, this affects the more subtle temperature-sensitive aspects of entrainment. A similar argument based on an interruption of the entrainment network can also be proposed to explain the corresponding results with norpAP41; cryb double mutants, because in this case per mRNA is assumed to be noncycling because of the cryb background. However, the locomotor behavior of cryb single mutants remains thermosensitive even though overall per mRNA is noncycling. Thus, only when the photoreceptive pathway and mRNA cycle are both compromised (as in gl and norpAP41; cryb) is locomotor behavior insensitive to temperature-dependent changes in per splicing levels (Collins, 2004).

A model is presented of how light and temperature may set the splicing level of the clock. How temperature is detected by the splicing machinery is not yet clear, but there is compelling evidence that norpA plays a role. At low temperatures, the splicing level is primarily set by light via the visual system rather than Cry, which is then remembered during the night. In longer periods of darkness such as in DD, this memory decays, and splicing levels begin to rise. Thus the visual system represses splicing by enhancing the effects of an unknown repressor molecule(s) that is sensitive to temperature change and the norpA PLC. At high temperatures, the regulation of splicing is more stringent and complex and recruits the circadian clock. Again, the light input received through the visual system sets the low splicing level during the day. This appears to also depend on the presence of at least two of the three molecules, Per, Tim, or Cry, because elimination of any one of these gives a barely detectable daytime rise in splicing, reflecting the very low levels of Per, Tim, and Cry at this time. However, elimination of both Per and Cry in the per01; cryb double mutant lifts all light-dependent repression during the day (Collins, 2004).

At night, the level of splicing set during the day by the visual system is again remembered and maintained by the clock at night. If per, tim, or cry is eliminated, then this repression of splicing is lost at night, generating the day/night difference in splicing levels. In gl60j cryb or norpAP41; cryb, because there is no visual light input during the day, there is no splicing level for the clock to remember, and therefore there is no day/night difference in splicing levels. Thus at high temperature, the visual system activates the repressor molecule during the day, and the clock maintains this activation at night. It is assumed that recruiting the clock at high temperature to inhibit per splicing is required to ensure that the fly's locomotor/foraging behavior is adaptive and does not encroach on those times of the day when there would be a significant risk of desiccation (Collins, 2004).

Disruption of Cryptochrome partially restores circadian rhythmicity to the arrhythmic period mutant of Drosophila

The Drosophila circadian clock is generated by interlocked feedback loops, and null mutations in core genes such as period and timeless generate behavioral arrhythmicity in constant darkness. In light-dark cycles, the elevation in locomotor activity that usually anticipates the light on or off signals is severely compromised in these mutants. Light transduction pathways mediated by the rhodopsins and the dedicated circadian blue light photoreceptor cryptochrome are also critical in providing the circadian clock with entraining light signals from the environment. The cryb mutation reduces the light sensitivity of the fly's clock, yet locomotor activity rhythms in constant darkness or light-dark cycles are relatively normal, because the rhodopsins compensate for the lack of cryptochrome function. Remarkably, when a period-null mutation was combined with cryb, circadian rhythmicity in locomotor behavior in light-dark cycles was restored, as measured by a number of different criteria. This effect was significantly reduced in timeless-null mutant backgrounds. Circadian rhythmicity in constant darkness was not restored, and Tim protein did not exhibit oscillations in level or localize to the nuclei of brain neurons known to be essential for circadian locomotor activity. Therefore, this study uncovered residual rhythmicity in the absence of period gene function that may be mediated by a previously undescribed period-independent role for timeless in the Drosophila circadian pacemaker. Although a molecular correlate for these apparently iconoclastic observations is not available, a systems explanation for these results is provided, based on differential sensitivities of subsets of circadian pacemaker neurons to light (Collins, 2005).

This study has revealed a surprising and intriguing restoration of circadian rhythmicity in LD cycles in per01; cryb flies. This partial rescue can even be extended to the adaptive thermal change in locomotor behavior mediated by 3' UTR splicing of the per transcript (Collins, 2004: Majercak, 2004; Majercak, 1997). A number of criteria have been used to dissect rhythmic behavior, including phase shifting in response to light pulses in LD and the use of T cycles to suggest that a residual oscillation, rather than an hourglass, underlies the behavior of the double mutant. The phase shifting of the per01; cryb oscillator is particularly informative because per01 is effectively rescuing this phenotype in cryb. This can be understood in terms of the robust, high-amplitude oscillator in cryb, being less 'perturbable' by light as Cry photoreception is lost, whereas the damped oscillator in per01; cryb is more sensitive to the environmental stimulus, precisely because of its low amplitude. The damped oscillation in the per01; cryb double mutant can be eliminated by removing tim function, but this is temperature dependent, so tim cannot supply the full explanation for these residual cycles. Although these experiments have focused on the 'evening' oscillator, of related interest is that the residual 'morning' oscillator that anticipates the lights-on signal in per01 was also observed. It is clear that both of these studies raise again the possibility of an underlying rhythmicity in per01 flies that was initially suggested from statistical analyses of mutant locomotor records (Collins, 2005).

The entrainment of a frequency-less oscillator in Neurospora crassa has been the subject of some recent debate, and the parallels with a residual rhythmicity in per-null Drosophila are striking. Furthermore, the rescue of per01 behavior by cryb would appear, at least superficially, to be similar to the situation in mammals in which a Cry mutation restores free-running rhythms to the arrhythmic mPer2 mutant mouse; this has been explained in terms of the freeing up in the double mutant of other mPer and Cry paralogues to interact and restore the original behavior. Since the fly does not have paralogues of per and cry, an explanation must be sought elsewhere. The only other genotypes identified so far with an anticipatory locomotor activity peak in LD and loss of rhythmicity in DD are disconnected (disco) and Pdf0. Neither mutation affects the molecular core of the circadian clock, rather the network of pacemaker neurons is disrupted. PDF is required for the functional integration of several clock neuronal groups within the brain, suggesting that disruption of interneuronal signaling causes arrhythmic behavioral output in the absence of synchronizing cues. In arrhythmic disco mutants, the clock gene expressing lateral neurons (LNvs and LNds) are usually absent, whereas the dorsal neurons are still present, thus indicating that the former are necessary for self-sustained rhythmicity, whereas the latter can only mediate rhythmic behavior under LD conditions (Collins, 2005).

This networking of clock neurons provides a basis for possible models to explain LD behavioral anticipation in the absence of Per, based on functional differences between the three groups of clock genes expressing LNs. Of these, only the small ventral LNs (sLNvs) and dorsal LNs (LNds) have a self-sustaining molecular clock when initially released into DD, although the latter depends on the former for synchronization. The third group, the large ventral LNs (l-LNvs) do not have a self-sustaining clock, although after a few days, tim mRNA again begins to accumulate rhythmically in these cells. Furthermore, rhythmic Tim expression is more sensitive to disruption by cry mutations in the l-LNvs, than in the s-LNvs or the LNds under LD conditions, suggesting that rhythmic output from the l-LNvs are compromised in a cryb background. In turn, this may contribute to the peculiar defects of cryb that includes robust entrainment to LD cycles, but significantly reduces behavioral phase shifts to brief light pulses, and, unlike wild-type, the maintenance of rhythmic behavior in constant light (Collins, 2005).

In the favored model, the robust s-LNv and LNd oscillators in cryb 'resist' the effects of brief light pulses, because of the impaired light input that is relayed to the s-LNvs, and from the s-LNvs to the LNds, by the more light-relevant l-LNvs. In per01, the molecular clock is severely dampened in all clock neurons, more so in the s-LNvs and LNds that have an endogenous cycle than the l-LNvs that do not. Thus, the light-mediated input from the l-LNv neurons into the s-LNvs, and indirectly to the LNds, is no longer resisted, and now overwhelms the residual damped per01 oscillator in these neurons, stimulating light-induced non-rhythmic locomotor behavioral output. However, when cryb and per01 are combined, the weak oscillator of per01 is no longer overcome by the light input because it is attenuated by cryb and mediated via the l-LNvs. Thus, rhythmic behavior is observed in LD cycles, providing a glimpse of the residual Per-independent, partly Tim-regulated clock. This model is preferred over a simpler one in which only the s-LNvs are involved, because previous studies have shown that the only direct photoreceptive input into these neurons is from the Hofbauer-Buchner eyelet, which is a very weak photoreceptor at best and it cannot, in the absence of other photoreceptors, entrain the fly's behavior (Helfrich-Forster, 2002). Thus, it is difficult to see how light information would be received by the s-LNvs to entrain the per01; cryb double mutant so effectively, unless it is transmitted from another neuronal source: the l-LNvs (Collins, 2005 and references therein).

In support of the model, there appears to be both direct and indirect neural connections between the compound eyes and the l-LNvs, suggesting that the l-LNvs may act as the light 'amplifier'. This study extends earlier observations by showing that photoreceptor cells expressing the rhodopsin genes, Rh3 and Rh5, send their axons through the medulla terminating in close proximity to the general region where the l-LNvs likely extend their dendritic arborizations..Although not definitive, these results support earlier claims that the photoreceptors may directly (or indirectly) synapse with the l-LNvs. As stated above, these molecular and proposed functional differences between s- and l-LNvs may also contribute to explaining the loss of light responsiveness in cryb mutant flies, which are blind to constant light and brief light pulses, despite retaining light input from the canonical visual transduction pathway. Thus Cryptochrome, aside from being a photoreceptor in its own right, also appears to control a gateway for rhodopsin-mediated light input into the clock (Collins, 2005).

Although the disruption of neural networks in this way probably explains the light responses of the clock in per01; cryb, it offers no molecular basis for the observed behavior. The loss of anticipation in tim-null-bearing genotypes suggests that Tim may play a key role. Although no significant nuclear Tim was observed in the LNvs or LNds of per01; cryb, the latter neurons being particularly relevant for providing the evening peak of locomotor activity present in the double mutants, it is suspected that Tim is shuttling continually in and out of the nucleus because Tim can enter the nucleus alone, but requires Per for nuclear retention, at least in larval clock neurons. Once in the nucleus, Tim is presumably interacting with as yet unidentified protein(s) in a light-dependent manner, generating behavioral rhythms in the double mutants. A microarray study found that 18 of the 72 genes that cycled in LD in wild-type also cycle in per01. Any one or more of these light-controlled proteins could therefore interact with Tim, contributing to the light-dependent oscillator of per01; cryb. In fact, it has been noted by others that a glutamine-rich transcriptional activator domain found within Tim may allow it to regulate other genes in a Per-independent manner (Collins, 2005).

Veela defines a molecular link between Cryptochrome and Timeless in the light-input pathway to Drosophila's circadian clock

Organisms use the daily cycles of light and darkness to synchronize their internal circadian clocks with the environment. Because they optimize physiological processes and behavior, properly synchronized circadian clocks are thought to be important for the overall fitness. In Drosophila, the circadian clock is synchronized with the natural environment by light-dependent degradation of the clock protein Timeless, mediated by the blue-light photoreceptor Cryptochrome (Cry). This paper report identification of a genetic variant, Veela, which severely disrupts this process, because these genetically altered flies maintain behavioral and molecular rhythmicity under constant-light conditions that usually stop the clock. The Veela strain carries a natural timeless allele (ls-tim), which encodes a less-light-sensitive form of Timeless in combination with a mutant variant of the F-box protein Jetlag. However, neither the ls-tim nor the jetlag genetic variant alone is sufficient to disrupt light input into the central pacemaker. A strong interaction between Veela and cryptochrome genetic variants, demonstrating that the Jetlag, Timeless, and Cry proteins function in the same pathway. Veela also reveals a function for the two natural variants of timeless, which differ in their sensitivity to light. In combination with the complex array of retinal and extraretinal photoreceptors known to signal light to the pacemaker, this previously undescribed molecular component of photic sensitivity mediated by the two Timeless proteins reveals that an unexpectedly rich complexity underlies modulation of this process (Peschel, 2006).

Veela is abnormally rhythmic in constant light, similar to mutations affecting the blue-light photoreceptor Cry. Veela's phenotype is due to the simultaneous presence of the ls-tim allele (encoding a less-sensitive form of Tim) and the jetc variant encoding a mutant form of the F-box protein Jet. Veela genetically and molecularly interacts with cryb, indicating that Tim, Jet, and Cry function in the same circadian light-synchronization pathway. These findings show that additional factors are necessary to elicit the phenotypes previously associated with jet variants. In particular, only when jetc is linked to the ls-tim allele, which encodes a less-light-sensitive form of Tim, can abnormal behavioral rhythmicity in LL be observed. The importance of the Jet protein per se in the light-entrainment process remains unclear, also when considering certain aspects of the original jet study in conjunction with the findings presented in his study. All control flies used by Koh (2006) came from a y w genetic background, which carries the s-tim allele. Contrarily, all jetc or jetr mutant flies carried the ls-tim allele (necessarily; otherwise, they would have behaved like WT). It follows that behavioral and molecular differences between control and mutant flies reported by Koh in fact reflect the combined effects of ls-tim (vs. s-tim) and jetc (vs. jet +). In conjunction with Western blot data showing an increased jet-independent stability of the larger Tim form compared with the smaller one, it seems that the effects on Tim degradation previously attributed to jet variants are mainly a reflection of the different features of the two Tim proteins. This may also explain why Koh saw only very subtle effects of their mutant Jet proteins on Tim degradation in vitro (Peschel, 2006).

Nevertheless, it is clear that jet influences the light-input pathway of the circadian clock; WT flies behave arrhythmically in LL, even though they carry ls-tim. Moreover, Veela strongly interacts with Cry, a crucial protein for circadian light input in flies. Importantly, these findings reveal that, with the current knowledge, an in vivo function for jet's F-box protein can be demonstrated only when the available jet variants are combined with ls-tim. To ultimately resolve the specific function of the Jet protein in the light-input pathway, loss-of-function jet mutants (Debruyne, 2006) or specific RNAi transgenics need to be generated and analyzed chronobiologically (Peschel, 2006).

Characterization of Veela also led to the assignment of a biological function for the two natural tim variants that were identified many years ago. This study has show that Tim encoded by the ls-tim allele is more stable after light exposure, and that this increased stability has behavioral consequences when flies are exposed to constant light; if the ls-tim allele is linked to jetc, these flies behave abnormally rhythmically in LL. If jetc is linked to s-tim, the flies behave like WT and become arrhythmic in LL. Therefore, the less-light-sensitive Tim form encoded by ls-tim is necessary and sufficient to block light input into the circadian clock of jetc flies. In nature, the natural polymorphism at the tim (and perhaps jet) locus might be used to fine-tune the light sensitivity of Drosophila's circadian clock on a purely molecular level. In conjunction with various anatomical light-input routes that are known to send light to Drosophila's circadian pacemaker, these findings reveal a glimpse of the potential complexity of this process. The frequent and random occurrence of tim and jet variants in currently used laboratory strains also speaks to a more cautious strain selection and genotyping in all studies concerning light-input pathways to the circadian clock (Peschel, 2006).

Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest-activity rhythms and relies upon different groups of PERIOD (Per)-expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional Cryptochrome (Cry), as opposed to wild-type flies, are rhythmic in constant light, transgenic flies were analyzed expressing Per or Cry in the LN-MO or LN-EO. Under constant light conditions and reduced Cry function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, this study shows that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. It is therefore proposed that day-night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons (Picot, 2007).

The PDF-expressing LNs and the PDF-negative LNs were previously characterized as morning and evening cells, respectively, in LD conditions. Furthermore, the morning LNs were able to drive robust 24-h rhythms in DD, whereas evening LNs were not. This study shows that in LL, the evening LNs drive robust rhythms when cryptochrome signaling is absent or reduced, whereas the morning cells are not able to do so. Surprisingly, the molecular oscillations of both groups can be uncoupled from behavioral rhythmicity, depending on light conditions. In DD, the two LN groups show autonomous molecular cycling, but there is no behavioral output when the LN-EO is cycling alone. In LL (and reduced Cry signaling), both groups still show autonomous cycling, but there is no behavioral output when the LN-MO is cycling alone. It is therefore concluded that light has opposite effects on the behavioral output of the two LN oscillators, activating it from the evening LNs and inhibiting it from the morning LNs (Picot, 2007).

The opposite effects of light on the behavioral outputs do not appear to be related to entrainment, since Per oscillations in both the PDF-positive and PDF-negative LNs are synchronized to the LD cycles even in the absence of Cry signaling. The inhibiting effect of light on the LN-MO behavioral output is abolished when the visual system is genetically ablated. This suggests that the projections of the visual system photoreceptors convey, not only input information to the PDF cells (light entrainment), but also signals to control their behavioral output (light inhibition). It is tempting to speculate that light exerts both effects through a direct connection of the PDF cells with the visual system. The Hofbauer-Büchner eyelet photoreceptors that project directly to the LN-MO neurons and participate in the entrainment provide a possible pathway (Picot, 2007).

It was recently reported that the overexpression of Per or of the Shaggy (Sgg) kinase in the PDF-negative clock neurons induced rhythmic behavior in LL. The rhythmicity was associated with the cycling of Per subcellular localization in some of the DNs, whereas the PDF-expressing cells were molecularly arrhythmic. These studies therefore concluded that some DN subsets are able to drive behavioral rhythms in LL. Different groups of PDF-negative cells may thus be able to drive behavioral rhythms in constant light, depending on whether and how the molecular clock is manipulated. Such manipulation could also directly affect molecular oscillations, making them less easy to detect. Since Cry does not appear to have a core clock function in the brain, these data are largely based on situations in which the clock mechanism is little if at all altered. The data support a major contribution of the LN-EO to the robust rhythms of cryb mutants in LL (Picot, 2007).

The strong rhythmicity of the cryb pdf0 double mutants in LL contrasts with their weak rhythmic behavior in DD. Altogether, these results strongly suggest that this robust rhythm is generated by the LN-EO, which would therefore behave as a PDF-independent autonomous oscillator. However, the period of the oscillator is clearly influenced by PDF signaling, and thus by the LN-MO, going from 24–25 h in cryb to 22–23 h in cryb pdf0 flies. An attractive possibility is that the strong short-period rhythm observed in the cryb pdf0 double mutant in LL has the same neuronal origin as the weak short-period rhythm described for pdf0 mutants in DD. The cellular basis of this PDF-independent oscillator in DD remains unclear, although the presence of similar rhythms in flies genetically ablated for the PDF-expressing neurons suggests that it originates from other clock cells (Picot, 2007).

Different results were obtained for the recently described DN-based LL oscillators. When transferred to a pdf0 background, all SGG-overexpressing flies were found to be arrhythmic, whereas about 60% of the Per-overexpressing flies displayed long-period rhythms. This suggests that different types of DNs with different sensitivity to PDF may have been analyzed in these two studies. Although some DNs may contribute to the PDF-independent rhythms, these data suggest a strong contribution of PDF-negative LNs to the rhythmic behavior that persists in pdf0 mutants. The weakness of the short-period rhythm of pdf0 flies in DD may reflect the inhibition of the LN-EO output in the absence of light (Picot, 2007).

These results indicate that whereas the LN-MO autonomously drives rhythmic behavior in constant darkness, the LN-EO plays this role in constant light, if Cry signaling is abolished or reduced. It is thus suggested that in natural LD conditions, Drosophila behavior could be driven by the LN-MO during the night, and by the LN-EO during the day, when cryptochrome is quickly degraded by light. This supports a model of a light-induced switch between the circadian oscillators of the LNs that would allow a better separation of the dawn and dusk activity peaks in day–night conditions. It has been shown that PDF-expressing LNs drive the clock neuronal network in short days, whereas PDF-negative DN subsets take the lead in long days. Thse results suggest that the PDF-negative cells of the LN-EO could also be a major player during the long days. Surprisingly, it was found that light does not seem to act on the molecular oscillations, but inhibits the LN-MO behavioral output and promotes the LN-EO behavioral output, which may provide an efficient fine tuning of the contributions of the two oscillators. It therefore appears that the visual system controls both the input (entrainment) and the behavioral output of the LN oscillators in the Drosophila brain clock. In species such the honeybee or the flour beetle, which appear to lack a light-sensitive Cry protein, this role of the visual system may be particularly important (Picot, 2007).

Identifying specific light inputs for each subgroup of brain clock neurons in Drosophila larvae

In Drosophila, opsin visual photopigments as well as blue-light-sensitive cryptochrome (Cry) contribute to the synchronization of circadian clocks. This study focused on the relatively simple larval brain, with nine clock neurons per hemisphere: five lateral neurons (LNs), four of which express the pigment-dispersing factor (PDF) neuropeptide, and two pairs of dorsal neurons (DN1s and DN2s). Cry is present only in the PDF-expressing LNs and the DN1s. The larval visual organ expresses only two rhodopsins (RH5 and RH6) and projects onto the LNs. PDF signaling is required for light to synchronize the Cry- larval DN2s. This study shows that, in the absence of functional Cry, synchronization of the DN1s also requires PDF, suggesting that these neurons have no direct connection with the visual system. In contrast, the fifth (PDF-) LN does not require the PDF-expressing cells to receive visual system inputs. All clock neurons are light-entrained by light-dark cycles in the rh52;cryb, rh61 cryb, and rh52;rh61 double mutants, whereas the triple mutant is circadianly blind. Thus, any one of the three photosensitive molecules is sufficient, and there is no other light input for the larval clock. Finally, it was shown that constant activation of the visual system can suppress molecular oscillations in the four PDF-expressing LNs, whereas, in the adult, this effect of constant light requires Cry. A surprising diversity and specificity of light input combinations thus exists even for this simple clock network (Klarsfeld, 2011).

The larval brain clock and its light inputs are generally considered much simpler than their adult counterparts. We find here that larvae, with only nine clock neurons and 12 photoreceptors on each side, nevertheless display four distinct combinations of light inputs (Klarsfeld, 2011).

Anatomical data and the present work show that PDF+ LNs are the only brain cells to perceive light both cell autonomously (via CRY) and through a direct connection to the visual system. They thus appear to be the main players responsible for synchronizing the larval brain clock network to LD cycles. The DN2s, in contrast, possess neither type of light input, but play a major role in the temperature entrainment of the clock (Picot, 2009). Previous studies have shown that the DN2s are intrinsically blind and must rely on PDF signaling from the LNs to synchronize to LD cycles (Picot, 2009). This study shows that the other dorsal group, the DN1s, is also sensitive to PDF signaling. In the absence of functional Cry, Pdf is required to synchronize DN1s by light, as demonstrated by the lack of Per oscillations in the DN1s of the cryb pdf0 double mutant. This is consistent with the presence of a dendritic-like arborization from the DN1s close to the dorsal projection of the LNs. On the other hand, it tends to exclude a functional connection between the DN1s and the larval visual system, in agreement with the absence of DN1 neurites reaching the Bolwig's nerve terminals (Klarsfeld, 2011).

The Pdf-dependent entrainment of both DN1s and DN2s by the visual system also indicates that the fifth LN, although projecting largely like the Pdf+ LNs, cannot synchronize the DNs. However, the fifth LN might be involved in RH5-dependent acute larval responses to light, which do not require the Pdf+ LNs. The entrainment of the fifth LN in the absence of both Cry and the Pdf+ LNs suggests a direct connection to the visual system, in agreement with its arborization in the larval optic neuropil. Recent single-cell analysis indeed revealed this arborization to be even broader than that of the Pdf+ LNs. However, such connection to the visual system does not allow constant light to disrupt Per oscillations in the fifth LN, contrary to the Pdf+ LNs, suggesting different downstream signaling in these two types of visual system targets. Finally, the results suggest a hitherto unsuspected connection between some Cry+ neurons and the fifth LN. This connection does not rely on Pdf and could be directly from the DN1s or the Pdf+ LNs, which both have projections in the vicinity of the fifth LN's projections (Klarsfeld, 2011).

More generally, the fact that the Cry- fifth LN and DN2s display normal Per oscillations in the absence of a functional visual system is consistent with Cry transmitting light information in a non-cell-autonomous way. This has already been proposed in the adult brain for the three Cry- dorsal lateral neurons and the DN2s. However, it remains possible that such nominally Cry- cells in the adult express very low levels of Cry, as judged from reporter gene expression. In contrast, Cry expression in the larval 5th LN and DN2s was observed neither with antibodies, not with any reporter lines. The present results make it even less likely, because constant light does not affect these neurons at all (Klarsfeld, 2011).

The role of the Pdf neuropeptide in the light entrainment of the DN1s and DN2s appears somewhat different for the two subgroups. First, Pdf sets the DN1s and DN2s to very different phases: the DN2s are set in antiphase with the LNs, whereas the DN1s are set in phase with the LNs. This suggests that the corresponding signaling cascades differ somewhere downstream from the Pdf receptor. In addition, the dispersion of cell labeling intensities suggests that unentrained DN2s oscillate, although asynchronously (even within a single brain hemisphere), while unentrained DN1s do not, but rather express constant, moderate Per and TIM levels. The same may hold true for completely blind larvae. While the LNs and DN2s seem to oscillate with random individual phases, all DN1s display very similar Per levels. This implies that, in LD, Pdf may be needed not only to synchronize but to trigger (or at least maintain until the third larval stage) DN1 oscillations in the absence of Cry activation. In contrast, Pdf synchronizes persistent autonomous oscillations in the DN2s. The non-autonomous cycling of the Cry-expressing DN1s suggests that they may have an important role in synchronizing the network to LD cycles. Conversely, the capacity of the DN2s for autonomous cycling in the absence of light cues may relate to their specific role in temperature entrainment (Klarsfeld, 2011).

Lack of entrainment by light was previously reported for the LNs and the DN1s in norpAP41;;cryb larvae, while, rather surprisingly, molecular oscillations were still detected in their DN2s. While the DN2s require Pdf to entrain in LD, they appear to entrain to temperature cycles very efficiently on their own (Picot, 2009). This means one cannot exclude the possibility that, in a previous study, small temperature changes induced by the LD cycles weakly entrained these neurons, but not the others. Alternatively, the DN2s might collect light information from a NORPA-independent pathway. NORPA-independent photoreception appears to participate in adult circadian photoreception (Klarsfeld, 2011).

The results show that RH5, RH6, and Cry are the only light input pathways for synchronizing the larval clock neurons to LD cycles. RH5, RH6, and Cry are each sufficient alone to entrain all these neurons, whereas, in the adult, some clock neurons fail to entrain in the absence of Cry. At least two more rhodopsins, including RH1 and a UV-blue one (RH3 and/or RH4), participate in the adult, so that all available rhodopsins in the adult eye may also be involved in entraining the clock. Recently, at least two classes of larval sensory neurons, outside BO, have been shown to express visual transduction components. One of these two is involved in thermal preferences, with RH1 as the presumed temperature sensor, while the other mediates rhodopsin-independent avoidance of very high light intensities. At least in the conditions used in this study, these novel sensory pathways do not seem to contribute to circadian light entrainment (Klarsfeld, 2011).

Interestingly, constant light, acting Cry independently through the visual system, can abolish or greatly disturb oscillations in the Pdf+ LNs of larvae but not adults. Similarly, the larval visual system is required for fast TIM degradation in the LNs at the end of the night. The Pdf+ LNs of eyeless adult flies, in contrast, seem to respond normally even to a very short light pulse, suggesting that the visual system is dispensable for the response to light pulses in adults, but not larvae. The different sensitivity of the larval clock to visual system inputs could be related to the change in signaling pathways that occurs as the larval cholinergic visual system develops into the adult histaminergic visual system. Moreover, contrary to the adult situation, the larval visual nerve may be light sensitive all along its length, down to its connection with the LNs, as judged from RH6 and NORPA expression. How visual system signaling ultimately affects the clock, whether in larvae or adults, remains to be discovered (Klarsfeld, 2011).

Both RH5+ and RH6+ BO photoreceptors contribute to the light responses of the larval brain clock that were tested, i.e., entrainment in LD and disruption of LN rythmicity in LL. Similarly, both photoreceptor types are equally able to suppress TIM levels in the LNs after a 2 h light exposure at the beginning of the night. In contrast, RH5 fibers alone specifically mediate acute larval responses to light, while RH6 fibers alone are specifically required for the development of a serotonergic arborization that also contacts the LNs. That RH6 activation strongly disrupts molecular oscillations in the LNs even in RR was, however, not anticipated. In the adult, RR does not affect molecular or activity rhythms (Klarsfeld, 2011).

Activation of RH6 above 600 nm is less than a few percent of peak activation at ~510 nm. This suggests that the clock of the larval LN is extremely sensitive to red light, which may explain why no larval activity rhythm was recorded in a study that used video tracking in constant red light. A strong sensitivity of larvae to the more penetrating, longer wavelengths of light may be related to their burrowing lifestyle (Klarsfeld, 2011).

Dopamine acts through Cryptochrome to promote acute arousal in Drosophila

The fruit fly, Drosophila melanogaster, is generally diurnal, but a few mutant strains, such as the circadian clock mutant ClkJrk, have been described as nocturnal. This study reports that increased nighttime activity of Clk mutants is mediated by high levels of the circadian photoreceptor Cryptochrome (Cry) in large ventral lateral neurons (l-LNvs). Cry expression is also required for nighttime activity in mutants that have high dopamine signaling. In fact, dopamine signaling is elevated in ClkJrk mutants and acts through Cry to promote the nocturnal activity of this mutant. Notably, dopamine and Cry are required for acute arousal upon sensory stimulation. Because dopamine signaling and Cry levels are typically high at night, this may explain why a chronic increase in levels of these molecules produces sustained nighttime activity. It is proposed that Cry has a distinct role in acute responses to sensory stimuli: (1) circadian responses to light, as previously reported, and (2) noncircadian effects on arousal, as shown in this study (Kumar, 2012).

Both dopamine and Cry are required for acute arousal at night. An arousal-promoting role for dopamine is supported by earlier studies. Dopamine transporter mutants were shown to exhibit a decreased arousal threshold, whereas the pale mutants exhibit an increased arousal threshold. This effect on arousal reflects a novel role for dopamine in sensory responses at night. Cry has not been implicated in arousal, although it promotes neural activity in a light-dependent manner. As in the case of the neural activity assay, this study found that arousal in response to sensory stimuli is reduced but not eliminated by the cryb mutant, indicating that the mechanism is distinct from the circadian response that is eliminated by cryb. Both neural activity and behavioral arousal responses are eliminated by the cry0 mutant, suggesting that the neural response underlies the behavioral effect. It is proposed that Cry is required at multiple levels for acute responses to sensory stimuli. In the case of circadian photoreception, it is absolutely required for phase-shifting in response to pulses of light, although not for entrainment to LD cycles. In the case of responses to sensory stimuli, again it is required for the startle response. Any effects of Cry on light-induced activity (physiological or behavioral) are likely to be acute, since Cry gets degraded with increased light treatment. Interestingly, in two different species of Bactrocera, cry mRNA levels are positively correlated with the timing of mating, which is also indicative of a regulated response required for a specific purpose. A chronic effect is seen only in the case of Drosophila Clk mutants, where levels of Cry are considerably higher than normal, and dopamine signaling is also elevated. It is hypothesized that Cry only promotes nocturnal activity in flies with chronically elevated dopamine signaling because dopamine acts as a trigger to activate Cry. However, this activation may be different from activation in a circadian context, given that different mechanisms appear to underlie the circadian and arousal-promoting roles of Cry. Dopamine- and Cry-mediated locomotor activity is restricted largely to the night because of light-induced Cry degradation and light-induced inhibition of dopamine signaling (Kumar, 2012).

At night, animals sleep, and the arousal threshold is increased. However, they still need to be able to respond in case of sudden events. It is speculated that dopamine and Cry are essential for this. In the case of Cry, it may arouse the animal and also reset the clock. For instance, the immediate response of an animal to a pulse of light at night is to wake up, which may be driven by the arousal-promoting role of Cry. In addition, the circadian clock must be reset, which requires the circadian function of Cry. Whether or not these roles of Cry are conserved, it is speculated that dopamine functions similarly in mammals. Interestingly, melanopsin, which is the circadian photoreceptor in mammals (analogous to Cry in flies), is regulated by dopamine in intrinsically photosensitive retinal ganglion cells (ipRGCs). Like Cry, melanopsin is also required for acute behavioral responses to light, specifically for sleep induction in nocturnal animals during the day. These ipRGCs have been proposed as functionally similar to l-LNvs, so a conserved function for the relevant molecules is intriguing. Finally, it is noted that elevated dopamine has been linked to increased nighttime activity in humans, which are, of course, diurnal like Drosophila. People with Sundown syndrome or nocturnal delirium show increased agitation and sleep disturbances in the early evening, which can be treated with anti-psychotic medications that target dopamine signaling (Kumar, 2012).

Phase-shifting the fruit fly clock without cryptochrome

The blue light photopigment cryptochrome (CRY) is thought to be the main circadian photoreceptor of Drosophila melanogaster. Nevertheless, entrainment to light-dark cycles is possible without functional CRY. This study monitored phase response curves of cry01 mutants and control flies to 1-hour 1000-lux light pulses. It was found that cry01 mutants phase-shift their activity rhythm in the subjective early morning and late evening, although with reduced magnitude. This phase-shifting capability is sufficient for the slowed entrainment of the mutants, indicating that the eyes contribute to the clock's light sensitivity around dawn and dusk. With longer light pulses (3 hours and 6 hours), wild-type flies show greatly enhanced magnitude of phase shift, but CRY-less flies seem impaired in the ability to integrate duration of the light pulse in a wild-type manner: Only 6-hour light pulses at circadian time 21 hours significantly increased the magnitude of phase advances in cry01 mutants. At circadian time 15 hours, the mutants exhibited phase advances instead of the expected delays (Kistenpfennig, 2012).

There is one main difference between wild-type and CRY-deficient flies regarding parametric light effects: cry mutants do not become arrhythmic at LL, not even at high irradiances. In this respect, the clock of CRY-deficient flies appears similar to that of mammals because the clock of most mammalian species runs under constant dim light. On the molecular level, this difference is easy to understand because light-activated Drosophila CRY leads to degradation of TIM. After TIM has disappeared, PER cannot be stabilized, and as a consequence, the clock stops. Indeed, it has been noted that after 6-hour light pulses, the activity rhythm of wild-type flies always started with the same phase, suggesting that the clock had completely stopped and was restarted after lights-off. Mammalian-like CRY is not light sensitive, and thus, light will probably not completely stop the mammalian clock, at least not after light pulses of 6 hours. Only a longer light exposure will stop the clock, as recently reported in mice after a pulse longer than 15 hours (Kistenpfennig, 2012).

The phase response curve for 12-hour light pulses shows that the clock of CRY-less flies is mainly light responsive at dawn and dusk. Such temporally restricted sensitivity must be sufficient for entrainment because dawn and dusk are the most important times at which a clock needs to respond to light. Because the light sensitivity of CRY-less flies is mediated by photoreceptor organs (as the compound eyes, the H-B eyelets, and possibly the ocelli), the current results suggest that these organs transmit photic information to the clock only in the morning and evening. Thus, different photoreceptors may be responsible for the different parts of a PRC (Kistenpfennig, 2012).

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

In nature, both daily light:dark cycles and temperature fluctuations are used by organisms to synchronize their endogenous time with the daily cycles of light and temperature. Proper synchronization is important for the overall fitness and wellbeing of animals and humans, and although a lot is known about light synchronization, this is not the case for 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. This study investigated whether different groups of the Drosophila clock neurons that regulate behavioral rhythmicity contribute to temperature synchronization at different absolute temperatures. Using spatially restricted expression of the clock gene period, this study shows that dorsally located clock neurons mainly mediate synchronization to higher (20°C:29°C) and ventral clock neurons to lower (16°C:25°C) temperature cycles. Molecularly, the blue-light photoreceptor Cryptochrome (Cry) dampens temperature-induced Period (Per)-Luciferase oscillations in dorsal clock neurons. Consistent with this finding, this study shows that in the absence of Cry very limited expression of Per in a few dorsal clock neurons is able to mediate behavioral temperature synchronization to high and low temperature cycles independent of light. This study shows that different subsets of clock neurons operate at high and low temperatures to mediate clock synchronization to temperature cycles, suggesting that temperature entrainment is not restricted to measuring the amplitude of such cycles. Cry dampens temperature input to the clock and thereby contributes to the integration of different Zeitgebers (Gentile, 2013).

This study has shown that different sets of clock neurons play a role for synchronization to low and high temperature cycles with identical amplitude. This shows that temperature entrainment does not solely rely on measurement of temperature differences but rather on measurement of absolute temperatures. This task is divided between different neuronal groups, opening the possibility that multiple temperature receptors -- expressed either in different clock neurons, other neurons in the brain, or in the PNS -- contribute to temperature entrainment. Cry seems to actively block the entrainment strength of temperature both at a molecular and behavioral level, which most likely contributes to Zeitgeber integration (Gentile, 2013).

Molecular evolution of a pervasive natural amino-acid substitution in Drosophila cryptochrome

Genetic variations in circadian clock genes may serve as molecular adaptations, allowing populations to adapt to local environments. This study carried out a survey of genetic variation in Drosophila cryptochrome (cry), the fly's dedicated circadian photoreceptor. An initial screen of 10 European cry alleles revealed substantial variation, including seven non-synonymous changes. The SNP frequency spectra and the excessive linkage disequilibrium in this locus suggested that this variation is maintained by natural selection. Focus was placed on a non-conservative SNP involving a leucine-histidine replacement (L232H); this polymorphism is common, with both alleles at intermediate frequencies across 27 populations surveyed in Europe, irrespective of latitude. Remarkably, it was possible to reproduce this natural observation in the laboratory using replicate population cages where the minor allele frequency was initially set to 10%. Within 20 generations, the two allelic variants converged to approximately equal frequencies. Further experiments using congenic strains, showed that this SNP has a phenotypic impact, with variants showing significantly different eclosion profiles. At the long term, these phase differences in eclosion may contribute to genetic differentiation among individuals, and shape the evolution of wild populations (Pegoraro, 2014).


EFFECTS OF MUTATION

A new rhythm mutation, cryb, has been isolated based on its elimination of period-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 proteins oscillate normally in temperature cycles). Consistent with the fact that light normally suppresses Tim protein, 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 (Stanewsky, 1998).

The cryb mutant does not exhibit phase shifts in response to light pulses. To assess clock resetting by brief pulses of light in (otherwise) constant darkness, phase response curves (PRC) were generated. Wild-type flies (and organisms in general) show phase delays after light pulses are given in the early subjective night, advances in late subjective night, and little or no phase shifting following pulses during the subjective day. When cryb flies are subjected to light pulses, no clear phase shifts result. This seems to contradict the fact that cryb flies tested for entrainment to different (phase shifted) LD cycles are able to "shift over" even at much lower light intensities. The apparent discrepancy could be explained by differences between the two experimental designs: in one case, flies are exposed to 12 hr of light, and in the PRC case, to only 10 min worth. In a very different kind of behavioral test involving responses to visual stimuli -- using short exposures of cryb flies to a relatively high light level, as in the PRC experiment -- the mutant exhibits normal optomotor behavior (Stanewsky, 1998).

The cry mutation does not eliminate cycling of Tim and Per protein levels within certain clock gene-expressing neurons. The cryb mutant exhibits rhythmic behavior in constant darkness in spite of the fact that no rhythmic protein expression during and after light entrainment is detectable. In Western blots involving head extracts, Per and Tim are measured mainly in photoreceptor cells (~90% of the anterior PNS and CNS cells expressing these genes. It was thought that rhythmic clock gene expression in the central pacemaker cells [the lateral neurons (LNs) that subserve behavioral rhythmicity (Kaneko, 1998)] could be masked by constitutive PER and TIM levels in the cryb mutant's eyes. The LNs consist of two groups of cells in each side of the brain, ~6 neurons in a relatively dorsal cluster (LNds) and ~10 such cells in a more ventrally located one (LNvs). Clock functions in the LNvs (along with the relevant molecular, physiological, and anatomical outputs) are probably sufficient to generate rhythmic behavior (Kaneko, 1998). Tim and Per expression were examined in the CNS (and in other cells of fly heads) by performing antibody stainings on sections of wild-type and cryb tissues. These were stained at two time points when Per and Tim each reached trough and peak levels. The staining intensities for different Per- and Tim-expressing cell types (compound-eye photoreceptors, glia, LNds, and LNvs) were scored blindly. Both proteins are observed to cycle in the LNs of cryb mutant flies, although with reduced amplitude as compared to wild type. Temporally constitutive, intermediate-level signals are observed in the eyes and glial cells, which explains the Western blot results obtained from extracts of cryb heads (Stanewsky, 1998).

The RNA oscillations observed for CRY mRNA (see cry Biological Overview) suggest that cry is a clock gene, and the primary sequence and light regulation indicate a role in photoreception. To link cry to circadian behavior, the GAL4 system was used to overexpress Cry in cells that govern locomotor activity rhythms. A newly generated tim promoter-GAL4 strain was crossed with a UAS-cry cDNA strain to generate progeny that should overexpress Cry in lateral neurons. Indeed, CRY mRNA levels are temporally constant and approximately 20-fold higher than the normal ZT1 peak level. The protein is also overexpressed (at least 30-fold at each time point) and cycles robustly, as expected from its light sensitivity. To quantitatively measure circadian light perception, flies were subjected to an anchored phase response curve (PRC) protocol; after entrainment to an LD cycle, flies were exposed to saturating or nonsaturating light pulses and the effect on behavioral phase measured. Control wild-type flies undergo a phase delay when the pulse is administrated during the early night and a phase advance during the late night. The overexpression had no significant effect on the period or strength of the locomotor activity rhythm, and there was no consistent or dramatic effect on the phase shift observed at high light intensities. However, the Cry overexpression strain is much more sensitive to light at low intensities. Especially in the delay zone, at ZT15, this effect is reproducibly very strong and suggests that Cry levels are normally limiting at low light intensities. In the advance zone, at ZT21, the magnitude of the effect is somewhat more variable from experiment to experiment. The striking light regulation of protein levels in clock-mutant as well as wild-type flies also indicates that Cry acts upstream of all known central pacemaker components (Emery, 1998).

Cryptochrome proteins are critical for circadian rhythms, but an understanding of their function(s) is uncertain. A mutation in Drosophila cryptochrome (dCRY) blocks an essential photoresponse of circadian rhythms, namely arrhythmicity under constant light conditions. This study concludes that dCRY acts as a key photoreceptor for circadian rhythms and that there is probably no other comparable photoreceptor in this species (Emery, 2000a).

Constant light causes the intrinsic circadian period of diurnal animals to shorten and that of nocturnal animals to lengthen (Aschoff's rule). More intense light produces more extreme effects, ultimately resulting in arrhythmicity in most mammals and birds. The circadian period of arthropods generally lengthens in constant light, whether the animal is nocturnal or diurnal. Drosophila melanogaster is no exception and intense constant illumination leads to arrhythmicity (Emery, 2000a).

The cryptochrome family includes blue-light photoreceptors. The single known Drosophila cryptochrome is thought to be a circadian photoreceptor: flies carrying a mutant allele, cryb, have severely decreased circadian photoresponses, whereas overproduction of dCRY causes increased photosensitivity. In mammals, however, mCRY1 and mCRY2 are more likely to be involved in the central clock mechanism (Emery, 2000a).

This raises the possibility that dCRY effects on photosensitivity reflect a role downstream of circadian photoreception, somewhere along the circadian light-input pathway or within the Drosophila central clock itself. This fits with the fact that cryb flies are still able to reset their circadian rhythm (entrain) to new light-dark cycles. cryb flies, however, remain behaviorally rhythmic in intense constant light, in contrast to wild-type flies and many other species that are arrhythmic under such conditions (Emery, 2000a).

The arrhythmicity of cryb flies must be a property of the cry gene, because the normal phenotype can be rescued by expressing wild-type dCRY in rhythm-generating cells of cryb flies. In intense constant light, the cryb mutant's behavior is strikingly similar to that of wild-type flies in constant darkness. An identical 24.7-hour period is also recorded under constant-darkness conditions, indicating that this slightly longer period is a characteristic of the background genotype. Thus, there is not even a detectable lengthening of period in the cryb mutant strain under constant light conditions. The free-running, circadian nature of cryb flies' constant-light behavior is further demonstrated by the 19.2-h period observed for flies with cryb in combination with a short allele of the period gene (per s) (Emery, 2000a).

These results show that the cryb mutation impairs the circadian photoreception pathway so profoundly that the fly cannot 'see' constant light. This mutant also responds very poorly to short light pulses; by these criteria, this circadian photoreceptor must be unique in Drosophila. How then can cryb flies entrain to different 24-h light-dark cycles? The missense cryb mutation might generate a protein with weak activity that would be sufficient for light-dark entrainment but not for a normal arrhythmic behavioral response to constant illumination. However, previous results suggest a different explanation: entrainment of cryb flies is through a second, completely separate light-input pathway. Visual photoreception may even directly influence locomotor activity, which then affects circadian rhythms only indirectly through a non-photic phase-resetting pathway (Emery, 2000a).

These results indicate that dCRY is an important circadian photoreceptor and probably the only dedicated one in Drosophila. Although additional central-clock functions for dCRY cannot yet be excluded, the abrogation of constant-light effects in cryb mutant flies indicates that this cryptochrome makes a unique contribution to Drosophila circadian photoreception (Emery, 2000a).

cryptochrome is an important clock gene. Recent data indicate that it encodes a critical circadian photoreceptor in Drosophila. A mutant allele, cryb, 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 cryb 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 pacemaker and a behavioral output system (Emery, 2000b).

The mutant cryb gene causes profound circadian light-response problems. While entrainment to 24 hr LD cycles still takes place in the mutant background, even this aspect of cryb circadian light perception is aberrant, because the mutant flies need much longer to entrain to a new cycle. A detailed examination of these LD entrainment data suggests that Cry contributes principally to adjusting the evening activity peak. The other major source of entrainment light information, the eyes, contributes principally to adjusting the morning peak. This fits with previous LD activity profile observations indicating that the phase of the evening activity peak is under clock control, whereas the phase of the morning peak is less sensitive to central clock mutations and is probably timed relative to some fixed environmental signal, e.g., the lights on or lights off transition. There is, however, a caveat, as the norpA; cryb double mutant phenotype suggests some interplay between the two light input pathways. The dual light input pathway for LD entrainment contrasts with the apparently unitary Cry input pathway for all other circadian photoresponses. This underscores the different nature of parametric (LD cycle) and nonparametric (short light pulse) entrainment. Taken together with the absence of any biological effect of CryB overexpression and the absence of CryB abundance cycling under LD conditions, cryb is probably a strong hypomorphic allele that encodes a protein without photoreceptor activity. This conclusion is consistent with the results of CryB expression studies in heterologous systems (Emery, 2000b and references therein).

Behavioral rescue experiments show that all known photoresponse defects of cryb are substantially rescued by expressing Cry only in the LNvs (ventral group of lateral neurons). The Cry-LNvs rescue is partial for the light pulse and constant light phenotypes, whereas it is almost complete for the LD entrainment defects of perS; cryb and norpAP41; cryb double mutant genotypes. In situ mRNA hybridization results suggest further that larvae also rely on Cry expression in the LNv precursor cells for circadian photoreception. There is no behavioral effect of Cry overexpression in the eyes, suggesting that the visual entrainment pathway is Cry independent (Emery, 2000b).

There are several possible explanations for the incomplete rescue of phase resetting and constant light arrhythmicity. pdf-GAL4 may be a relatively weak driver, such that Cry expression does not reach a required threshold level for full rescue. Cry may play a developmental role that is not fulfilled with pdf-GAL4-driven expression. There may also be other Cry-relevant cells, in addition to the LNvs, that contribute to circadian behavior. Consistent with this view, disco mutant flies lack LNvs but stay rhythmic for 1-3 days in DD. Cry expression studies should identify these accessory pacemaker cells. Good candidates are the dorsal lateral neurons and the dorsal neurons, both of which express Per and Tim and send projections to the same region of the brain as the LNvs (Emery, 2000b).

Why does Cry expression seem to be so high in LNvs, compared with other tissues, like the eyes? One possibility is that these neurons, located deep inside the brain, need to express high Cry levels to detect low light intensities. For example, this would allow the clock to respond at dawn and adjust its phase every day. Another explanation, more provocative perhaps, is that a high Cry concentration contributes to special pacemaker cell properties of the LNvs. In cryb, only the LNvs manifest Tim and Per cycling. This might reflect the high Cry levels in these cells, as well as a second, nonphotoreceptor contribution of Cry to pacemaker function. This Cry dark function could be to maintain the circadian oscillations of the molecular pacemaker, e.g., by contributing directly to the negative feedback loop, as shown in mammals. A true cry null mutation might therefore result in arrhythmicity, as observed in mammals (Emery, 2000b).

The evidence suggests that Cry contributes in a cell-autonomous manner to the Per/Tim molecular cycles. This presumably reflects independent photoreception of cells and tissues. In the periphery, Cry is absolutely required for light-dependent Tim degradation. Per, Tim, and Cry colocalization is not yet documented, but several studies have shown that cry is expressed in the body, as well as in different fly organs that contain autonomous clocks (Emery, 2000b).

Evidence is accumulating that the cell-autonomous property of circadian rhythms is universal, but with interesting differences between systems. The neuro-hormonal regulation of physiology may limit the autonomy of peripheral oscillators in mammals, where the suprachiasmatic nucleus (SCN) appears to be the principal central mammalian pacemaker organ. But the SCN, as well as most internal clocks and tissues, is probably not directly light sensitive. It receives photic cues from the eyes, where the circadian photoreceptor molecule has not been identified. Moreover, it is unclear whether mammalian Crys ever function as cell-autonomous photoreceptors, e.g., in cultured retina cells that exhibit circadian oscillations (Emery, 2000b).

Mammalian peripheral oscillators are probably under SCN control, largely through humoral connectors. This scheme accounts for the 4 hr phase difference between peripheral and SCN molecular cycles in vivo. The lack of any reported phase difference in Drosophila, i.e., between the molecular cycles in the periphery and LNvs, is consistent with rescue experiments and presumably reflects independent cell-autonomous connections by Cry to environmental light cues. Remarkably, this includes even the LNvs, since Cry expression within these brain pacemaker cells controls every known aspect of circadian behavioral photosensitivity. Although there is evidence in other systems for deep brain photoreceptors, Cry is the only functionally identified light sensor of this kind. Taken together with the recent identification of a behavioral output factor within the LNvs, they are now known to contain all three components of a functional, cell-autonomous circadian clock: photoreception, a central pacemaker, and well-defined output (Emery, 2000b).

It is concluded that all three clock components are present in the Drosophila circadian pacemaker cells. To control behavioral locomotor activity in a circadian manner, three elements are required: an input pathway, a pacemaker, and an output pathway. All three are present in the LNvs. By expressing Cry, the LNvs are directly sensitive to light. Cry controls the phase of the pacemaker, which is composed of a transcriptional feedback loop involving Per/Tim and Clock/Cyc dimers. The former regulates the transactivation potential of the latter in the nucleus. Doubletime, a kinase, is also necessary for central pacemaker function. This transcriptional loop regulates, through poorly understood mechanisms that may involve Vrille, the expression and the release of the neuropeptide PDF, which is an important output element of this circadian system (Emery, 2000b).

Cryptochromes are flavin/pterin-containing proteins that are involved in circadian clock function in Drosophila and mice. In mice, the cryptochromes Cry1 and Cry2 are integral components of the circadian oscillator within the brain and contribute to circadian photoreception in the retina. In Drosophila, cryptochrome (CRY) acts as a photoreceptor that mediates light input to circadian oscillators in both brain and peripheral tissue. A Drosophila cry mutant, cryb, leaves circadian oscillator function intact in central circadian pacemaker neurons but renders peripheral circadian oscillators largely arrhythmic. Although this arrhythmicity could be caused by a loss of light entrainment, it is also consistent with a role for Cry in the oscillator. A peripheral oscillator drives circadian olfactory responses in Drosophila antennae. Cry contributes to oscillator function and physiological output rhythms in the antenna during and after entrainment to light-dark cycles and after photic input is eliminated by entraining flies to temperature cycles. These results demonstrate a photoreceptor-independent role for Cry in the periphery and imply fundamental differences between central and peripheral oscillator mechanisms in Drosophila (Krishnan, 2001).

Circadian rhythms can be entrained by light to follow the daily solar cycle. In adult flies a pair of extraretinal eyelets expressing immunoreactivity to Rhodopsin 6 each contains four photoreceptors located beneath the posterior margin of the compound eye. Their axons project to the region of the pacemaker center in the brain with a trajectory resembling that of Bolwig's organ, the visual organ of the larva. A lacZ reporter line driven by an upstream fragment of the developmental gap gene Kruppel is a specific enhancer element for Bolwig's organ. Expression of immunoreactivity to the product of lacZ in Bolwig's organ persists through pupal metamorphosis and survives in the adult eyelet. It is thus demonstrated that the adult eyelet derives from the 12 photoreceptors of Bolwig's organ, which entrain circadian rhythmicity in the larva. Double labeling with anti-pigment-dispersing hormone shows that the terminals of Bolwig's nerve differentiate during metamorphosis in close temporal and spatial relationship to the ventral lateral neurons (LNv), which are essential to express circadian rhythmicity in the adult. Bolwig's organ also expresses immunoreactivity to Rhodopsin 6, which thus continues to be expressed in the adult eyelet. Action spectra of entrainment were compared in different fly strains: in flies lacking compound eyes but retaining the adult eyelet (so1), lacking both compound eyes and the adult eyelet (so1;gl60j), and retaining the adult eyelet but lacking compound eyes as well as Cryptochrome (so1;cryb). Responses to phase shifts suggest that, in the absence of compound eyes, the eyelet together with Cryptochrome mainly mediates phase delays. Thus a functional role in circadian entrainment first found in Bolwig's organ in the larva is retained in the eyelet, the adult remnant of Bolwig's organ, even in the face of metamorphic restructuring (Helfrich-Forster, 2002).

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. 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 glass60j;cryb double mutants, which lack all known external and internal eye structures in addition to being devoid of cryptochrome (Helfrich-Forster, 2001).

Extraocular photoreception is involved in the circadian systems of many organisms, but in most of them the structures and molecules involved are unknown or barely characterized. The present study demonstrates that nonretinal photoreceptors are involved in entrainment of Drosophila's circadian clock: fruit flies utilize at least a tripartite light-input pathway -- one pathway makes use of cryptochrome; a second acts through norpA-dependent photoreceptors in the compound eyes (perhaps also the ocelli), and a third pathway, which acts independently of norpA and cry gene functions, seems likely to involve the extraretinal Hofbauer-Buchner eyelets or other extraocular photoreceptors located in the brain (Helfrich-Forster, 2001).

These separate clock-input pathways influence the circadian system in different ways. Light input through CRY mainly entrains the evening peak of behavioral activity. Retinal and extraretinal eye structures predominantly synchronize the morning peak of activity. In spite of their different effects on behavioral rhythmicity, each light-input pathway alone seems capable of entraining the locomotor rhythm in a rather normal manner when the other one is impaired. Only when all three input routes -- those subserved by CRY, the compound eyes/ ocelli, and extraretinal eye structures are absent is the circadian system of the fly unable to respond to light. This is so at the cellular and at the behavioral level, thus revealing that fruit flies entrainment to multiple light-input pathways for adapting their circadian clock to the cyclic environmental LD changes. Interestingly, similar findings have recently been described for mice, for which depletion of retinal photoreceptors results in almost complete circadian blindness (Helfrich-Forster, 2001).

How Drosophila's multiple photoreceptors might interact remains a mystery but is likely related to the task, faced by many organisms, of extracting time-of-day information from dawn and dusk. During natural twilight, the quality of light changes in three important respects: the amount of light, its spectral composition, and the direction of incoming light (i.e., the position of the sun). These photic parameters all change in a systematic way at twilight times ['Heavenly shades of night are falling, it’s twilight time' (The Platters, 1958)]; all could be used by the circadian system throughout times of changing photic conditions at dawn and dusk, thus forming a versatile input system that subserves daily adjustments of the rhythm’s pace. Using different rhodopsins in addition to CRY permits the fly systems to scan all the way from the UV into the red (Helfrich-Forster, 2001).

The observation that the different pathways have at least some overlapping cellular targets provides a first hint about how the fly's different photoreceptors communicate: PER cycling in the s-LNv brain cells can be at the very least a tripartite light-input pathway -- one of them makes use of cryptochrome; a second pathway acts through internal eye structures (in the glass mutant). Similarly, in the absence of CRY and functional external eyes (in the norpAP41;cryb double mutant), the same neurons are acts independently of norpA and cry gene functions, synchronized by extraretinal photoreceptors. Such multiple input aimed at a single cell type would allow the organism to integrate the incoming light (Helfrich-Forster, 2001).

Among the multiple photoreceptors that could contribute to circadian photoreception in Drosophila, the contributions of the external eyes and of CRY function have been demonstrated with the help of the norpAP41;cryb doubly mutant flies that show entrainment defects, which are much more severe than those exhibited by either mutant alone. Nevertheless, this double-mutant type is not completely blind in the circadian sense, compared with the effects of the gl60j;cryb combination revealed in this study. Most norpAP41;cryb individuals are still able to entrain to LD cycles (Helfrich-Forster, 2001).

There are at least two possible explanations for these findings: cryb is not a loss-of-function mutation (indeed, this is a missense mutant), or other norpA-independent photoreceptors feed into the clock. The fact that flies could be generated that are totally circadian blind favors the second hypothesis (Helfrich-Forster, 2001).

The H-B eyelets send their projections directly to the accessory medulla and are therefore anatomically well suited to transmit light signals to the LNv pacemaker cells. The fact that, in the absence of CRY, PER cycling in the s-LN cells is nicely entrained also favors this hypothesis. The H-B eyelets express the photopigment Rhodopsin 6 and thus seem to have photoreceptive properties. The input pathway via Rhodopsin 6 might utilize the fly’s norpA-independent phospholipase C (which is expressed in many neurons in its phototransduction cascade (Helfrich-Forster, 2001).

Further candidates for circadian photoreceptors (revealed in this study) are the clock-gene-expressing dorsal neurons called DN1 cells. Like the photoreceptor cells of the compound eyes, the ocelli and the H-B eyelets, the DN1s appear to be eliminated by the gl60j mutation. Similar to the H-B eyelets, the DN1s send axonal projections toward the LNvs and could entrain the latter through this anatomical pathway. Furthermore, disconnected mutant flies, which largely lack the LNv but not the DN1 cells, are able to entrain to LD cycles. However, it is not known whether the DN1s express a photopigment or have photoreceptive properties like those exhibited by the H-B eyelets (Helfrich-Forster, 2001).

In spite of the inability of the gl60j;cryb double mutant to entrain to LD cycles, the behavior of such flies was still modified by the altered environmental conditions. This modulation of the activity level is interpreted as direct effects of light/radiant energy on locomotion that bypass the circadian system. Light-related energy often exerts such direct (or masking) effects on physiological parameters, including behavior. There are possibilities to distinguish real entrainment from masking: (1) after a phase shift of the LD cycle, the circadian rhythm often takes several transient cycles to reentrain to the new LD regime, whereas masking follows the new light schedule immediately (in Drosophila, transients can be observed at very low light intensities or in photoreceptor mutants like cryb; (2) after transfer into constant darkness, masking disappears immediately whereas an entrained rhythm starts to free run from the phase it had established in LD, and (3) masking is independent of a functional circadian clock -- for example, it occurs in animals deprived of their clock, such as squirrel monkeys suffering from lesions of their suprachiasmatic nucleus arrhythmic per0 mutants of Drosophila (Helfrich-Forster, 2001).

In gl60j;cryb, prominent masking is observed at the highest light intensities employed (1000 lux). The sudden increase of the activity level after lights off and phase delay of the LD cycle did not show any transients after the 8 hr phase shift. Furthermore, this apparently forced activity disappears immediately after transfer into DD and independent of a functional clock, owing to its presence in these doubly mutant flies, which exhibit apparent rhythmicity in constant darkness. Moreover, no entrained cycling of clock protein levels was observed in these gl60j;cryb flies, demonstrating that the forced behavior is neither the consequence of molecular clock gene cyclings nor a nonphotic Zeitgeber that could influence the circadian clock (Helfrich-Forster, 2001).

In summary, the circadian blindness of flies expressing both glass and cryptochrome mutations is due to elimination of all photoreceptor cells that participate in entraining the circadian system. Similar complex light entrainment pathways may also exist in vertebrates. Interestingly, cryptochromes, certain opsins located in the retina, and standard photoreceptor cells are candidates for participating in the circadian photoreception of mammals. Thus, rather than having an exclusive photopigment for entrainment of circadian rhythms, the situation in mammals could be similar to that in Drosophila: multiple photoreceptors share the workload involved in transmitting the principle environmental Zeitgeber to the circadian clock (Helfrich-Forster, 2001).

Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait

Identifying the genes involved in polygenic traits has been difficult. In the 1950s and 1960s, laboratory selection experiments for extreme geotaxic behavior in fruit flies established for the first time that a complex behavioral trait has a genetic basis. But the specific genes responsible for the behavior have never been identified using this classical model. To identify the individual genes involved in geotaxic response, cDNA microarrays were used to identify candidate genes and fly lines mutant in these genes were assessed for behavioral confirmation. The identities of several genes that contribute to the complex, polygenic behavior of geotaxis have thus been determined (Toma, 2002).

Pioneering experiments on Drosophila melanogaster and Drosophila pseudoobscura investigated the nature of the genetic basis for extreme, selected geotaxic behavior. These experiments constituted the first attempt at the genetic analysis of a behavior. Selection and chromosomal substitution experiments successfully showed that there is a genetic basis for extreme geotaxic response in flies and, by implication, for behavior in general. These experiments also added to understanding of the role of variation in phenotypic evolution and selection. Despite their seminal contributions in behavioral genetics, population genetics and the study of selection, by their nature these experiments could not identify specific genes (Toma, 2002 and references therein).

These results highlight both the success and the limitation of behavioral selection experiments. Although selection results tend to be representative of the natural interactions of genes that produce behavior and can demonstrate that a trait has a genetic basis, they do not pinpoint specific genes that influence the trait. This is partly due to the involvement of many genes and the relatively minor role of each in complex polygenic phenotypes -- a problem that is especially acute for the intrinsically more variable phenotypes that are associated with behavior. The advent of cDNA microarray technology offers an easily generalized strategy for detecting gene expression differences and can complement other means of identifying the genes that underlie complex traits. An expression difference may occur in a gene that is not itself polymorphic, but that gene may contribute to the realization of the phenotypic difference (Toma, 2002).

As a starting point for identifying genes that affect a complex trait, the selected, established Hi5 and Lo extreme geotaxic lines were examined for changes in gene expression between strains of Drosophila melanogaster subjected to long-term selection and isolation. A two-step approach was used: (1) the differential expression levels of mRNAs isolated from the heads of Hi5 and Lo flies was determined using cDNA microarrays and real-time quantitative PCR (qPCR); (2) a subset of the differentially expressed genes was independently tested for their influence on geotaxis behavior by running mutants for these genes through a geotaxis maze. It was reasoned that some of the differences in gene expression between strains might be related to phenotypic differences and that it should therefore be possible, at least in part, to reconstruct the phenotype with independently derived mutations in some of the differentially expressed genes (Toma, 2002).

The findings indicate that differences in gene expression can be used to identify phenotypically relevant genes, even when no large, single-gene effects are detectable by classical, quantitative genetic analysis. Three of the four genes implicated by microarray and qPCR measurements caused differences in geotaxis, whereas none of the six control genes had an effect. Only those genes that had larger differences in expression according to the microarrays, or that were significantly different according to qPCR results (cry, Pdf and Pen), significantly changed geotaxis scores. The converse was not true, because altered geotaxis behavior did not always accompany larger differences in mRNA levels, as shown by pros, although this might reflect the sensitivity of pros to aspects of the genetic context. All of the genes tested for which there was little or no difference in mRNA levels between the selected Hi5 and Lo lines also showed no influence on geotaxis behavior (Toma, 2002).

The directionality of behavioral and mRNA differences proved to be consistent with predictions that were based on expression levels. Homozygous null mutants of Pdf and cry showed a significant increase in geotaxis score, which is consistent with a lower level of expression of these genes in Hi5 relative to Lo. Similarly, the heterozygous Pen mutant showed a significant downward shift in geotaxis score, which is consistent with a lower level of Pen expression in Lo relative to Hi5. Thus, the change in behavior of the tested mutants corresponds to the direction predicted by differences in transcript level in the selected Hi5 and Lo lines (Toma, 2002).

Whereas the cry, Pen and female Pdf mutants produced the anticipated effect on behavior, the magnitude of behavioral effect was smaller than in the original selected lines. This probably reflects the difference between the aggregate effect of an ensemble of genes in the selected lines as opposed to the individual effect of a single mutant gene in a neutral background. In addition, their relatively small effects are exactly the results that one would predict in a polygenic system such as geotaxis behavior, in which many genes have small contributions to the overall phenotype. The three genes identified in this study would not have been predicted on the basis of their previously defined functions (Toma, 2002).

These results show that the two separate approaches to behavioral genetics -- the classical Hirschian quantitative analysis of genetic architecture and the modern Benzerian approach of single-gene mutant analysis -- are complementary and can be unified. This study used the results of a Hirschian approach of laboratory selection for natural variants to identify single gene differences, such as one would find in a Benzerian approach. The results are consistent with the suggestion that naturally occurring variants in behavior correspond to mild lesions in pleiotropic genes (Toma, 2002).

Finally, the results show that differences in gene expression identified by cDNA microarray analysis can be used as a starting point for narrowing down the numbers of candidate genes involved in complex genetic processes. Such an approach is analogous, as well as complementary, to the current method of mapping quantitative trait loci to large chromosomal intervals and then making educated guesses about which genes within those intervals may be involved in the trait (Toma, 2002).

The combination of selection, with its ability to exaggerate natural phenotypic variation, and global analysis of differences in gene expression by cDNA microarray analysis offers a promising approach to previously intractable molecular analyses of behavior. The geotaxis genes that were identified might have been the direct targets of selection, or they might be downstream of the direct targets. Additional studies using the Hi5 and Lo selected lines will be required to distinguish between these possibilities and to determine the causal role that these genes have in the context of the selected lines (Toma, 2002).

This study has gone from the selection of a 'laboratory-evolved' behavioral phenotype, to screening for mRNA differences, to partially reconstituting the phenotype using mutants. This shows the feasibility of combining genomic and classical genetic approaches for the breakdown and partial reassembly of an artificially selected behavioral trait (Toma, 2002).

Effects of combining a cryptochrome mutation with other visual-system variants on entrainment of locomotor and adult-emergence rhythms in Drosophila

Photoreception is an important component of rhythm systems and is involved in adjusting circadian clocks to photic features of daily cycles. In Drosophila, it has been suggested that there are three light input pathways to the clock that underlie rhythms of adult behavior: One involves the eyes; the other two involve extraocular photoreception through a structure called the Hofbauer-Buchner (H-B) eyelet and light reception carried out by pacemaker neurons themselves, mediated by a substance called cryptochrome. All photoreceptor cells including the H-B eyelet have been surmised to be removed by glass-null mutations. Mutations in the no-receptor-potential-A (norpA) gene cause the compound eyes and ocelli to be non-functional and may also affect the eyelet's function. The one cryptochrome mutant known (cryb) harbors an amino-acid substitution in the blue-light absorbing protein encoded by this gene. With regard to adult locomotor rhythms, all single mutants (gl60j, norpAP41, and cryb) re-entrained to altered light:dark (LD) cycles in which the L phase involves relatively intense light. Dropping light levels ca. 10 or ca. 30-fold permits small percentages of doubly-mutant gl60j;cryb flies clearly to re-synchronize their behavior. The marginal re-entrainability in the lowest-light situation nevertheless involves superior responsiveness of the gl60j;cryb type, compared with that observed using a different re-entrainment protocol. Furthermore, transgenic types in which rhodopsin-expressing cells within the H-B eyelet are ablated or suffer from the effects of tetanus-toxin also entrain with behavior similar or superior to that of gl60j;cryb at a low light level. Light inputs that are necessary to synchronize periodic adult emergence can be inferred to involve a cry-dependent pathway and perhaps also a norpA-dependent one, so that combining mutations in these two genes would cause cultures to be unentrainable. The current results show that each singly-mutant type ecloses rhythmically; flies emerging from norpAP41;cryb cultures also (on balance) exhibit solid eclosion rhythmicity. The ensemble of these behavioral and adult-emergence results suggest that additional light-to-clock pathways function within the system; alternatively, that rhythm assays employed in this study have teased out residual function of the mutated Cru protein (Mealey-Ferrara, 2003).

Seasonal behavior in Drosophila melanogaster requires the photoreceptors, the circadian clock, and phospholipase C

Drosophila locomotor activity responds to different seasonal conditions by thermosensitive regulation of splicing of a 3' intron in the period mRNA transcript. The control of locomotor patterns by this mechanism is primarily light-dependent at low temperatures. At warmer temperatures, when it is vitally important for the fly to avoid midday desiccation, more stringent regulation of splicing is observed, requiring the light input received through the visual system during the day and the circadian clock at night. During the course of this study, it was observed that a mutation in the no-receptor-potential-A(P41) (norpA(P41)) gene, which encodes phospholipase-C, generates an extremely high level of 3' splicing. This cannot be explained simply by the mutation's effect on the visual pathway and suggests that norpA(P41) is directly involved in thermosensitivity (Collins, 2004).

The proportion of per transcripts that were spliced at 18°C and 29°C, averaged over several LD 12:12 cycles was examined in Canton-S WT and per01, tim01, cryb, and per01; cryb mutant backgrounds. In all backgrounds splicing levels fall as the temperature rises, with 40%-60% of transcripts spliced at 18°C and 20%-45% at 29°C. However, not all genotypes react in the same way to temperature changes (Collins, 2004).

The smallest but nevertheless significant effect of temperature on splicing levels is observed in per01; cryb, suggesting that the temperature-sensing system for splicing may be compromised in the double mutant. A significant temperature x time effect reveals that the temporal patterns of cycling differ among temperatures, and the absence of any other significant interactions suggests that all genotypes respond similarly. There is very little evidence for a significant day/night cycle in the proportion of per transcripts that are spliced at 18°C, but at 29°C, all genotypes reveal a higher level of splicing post lights off (ZT12) compared to the trough at ZT8. At 18°C, the per01 and tim01 mutations have no significant effect on the level of splicing of per mRNA compared to WT. However in cryb flies, splicing levels are significantly elevated, particularly after lights off. This is also the case when per01; cryb is compared to WT. At 29°C, splicing levels are generally 5%-10% higher in per01, tim01, and cryb mutants compared to WT in the light, but 15%-20% higher after lights off at ZT12. This suggests that in the presence of light, splicing levels are reduced due largely to a clock-independent mechanism. In darkness, the clock and Cry become critical for maintaining this low splicing level at high temperatures (Collins, 2004).

The double mutant per01; cryb shows a highly significant increase in splicing of ~20% throughout the day/night cycle compared to WT. Thus, at high temperature, either the presence of the circadian photoreceptor Cry or a functional circadian clock is sufficient to largely repress daytime splicing. With both eliminated, daytime splicing levels are elevated. In contrast, repression of splicing in the absence of light requires the circadian clock plus Cry. It seems somewhat counterintuitive that Cry, which is activated by light, plays a more prominent role in repressing splicing at night than it does during the day (Collins, 2004).

Cry is likely to be a dedicated circadian photoreceptor yet at 29°C, splicing is repressed during the light phase even in cryb. This suggests that the light input to the splicing machinery cannot be primarily mediated by Cry. To confirm that light represses splicing, the effects that short photoperiods and constant darkness (DD) have on splicing levels in WT was investigated. There is a significant effect of reducing photoperiod on the splicing level with an elevated level of splicing in DD compared to LD 12:12, and similarly in LD 6:18, splicing levels are enhanced. Because the repression of splicing by light in LD 12:12 at 29°C does not require the presence of Cry, whether the visual system plays a role in setting the splicing level was investigated by examining the splicing of per transcripts in the mutants gl60j and norpAP41 (Collins, 2004).

The proportion of per mRNA transcripts that are spliced at both temperatures is increased in both the norpAP41 and gl60j backgrounds compared to WT. At 18°C, ~65% of per transcripts are spliced in norpAP41 and ~60% in gl60j, whereas at 29°C, these levels fall to ~55% and ~40%, respectively. Apart from a marginal difference between norpAP41; cryb, and norpAP41 at 18°C, there are no significant effects for either norpAP41 or gl60j when combined with cryb. These results indicate that the visual system rather than Cry is primarily responsible for the light-dependent repression of splicing. Unlike WT, per splicing levels do not rise after lights off at 29°C in either norpAP41 or gl60j (Collins, 2004).

Interestingly, in gl60j, there is a 20% difference between the splicing levels at different temperatures (60%-40%), whereas in norpAP41, this difference is reduced to 10% (65%-55%). The difference in gl60j is similar to that seen in WT (45%-25%). Thus per splicing in norpAP41 is relatively insensitive to temperature changes. It is also clear that the level of splicing in norpAP41 is significantly higher at all times and temperatures than gl60j. Therefore, the effect of norpAP41 on splicing is greater than that of gl60j, despite gl60j being the more severe visual mutant (Collins, 2004).

Locomotor activity profiles of all genotypes were also monitored at 18°C and 29°C. Because each genotype shows a higher level of splicing at 18°C than at 29°C, it would be predicted that this would generate an earlier evening activity peak at 18°C. This is the case for WT, cryb and norpAP41, but not for norpAP41; cryb or gl60j, where despite elevated splicing levels at higher temperatures, there is no difference in the phase of activity. gl60j cryb does not entrain to LD cycles at 25°C, so was not included in this analysis (Collins, 2004).

The average proportion of per transcripts that are spliced at 18°C rises from WT (45%) to cryb (50%) to gl60j (60%) to norpAP41 (65%), and at 29°C from WT (25%) to gl60j and cryb (~35%) to norpAP41 (55%). If the per splicing level is the only determinant of evening locomotor peak position, then a similar progression in the timing of this peak would be expected. The evening activity peaks of these different genotypes at 18°C and 29°C were compared. For norpAP41, cryb, and WT, there is an inverse relationship between average splicing levels and the position of the activity peak at 18°C, with norpAP41 and cryb having similarly advanced activity peaks compared to WT. At 29°C, the same inverse relationship holds, with norpAP41 advanced compared to cryb, which is in turn earlier than WT. Thus, those genotypes that show temperature-dependent changes in their evening activity generally display a correlation between average per splicing levels and the timing of the evening activity peak of the following day. Conversely, norpAP41; cryb and gl60j,, which show no significant differences in the phase of evening activity at different temperatures, have high splicing levels but relatively delayed evening activity peaks (Collins, 2004).

These observations raise the question of why the splicing level does not always relate to the timing of the evening locomotor activity peak, as in gl60j and norpAP41; cryb. Thus the per RNA profiles of gl60j and norpAP41 were compared to WT. Because per does not cycle in cryb whole head homogenates, the underlying cycle in this background was not examined. WT and norpAP41 show similar profiles, with an earlier per mRNA peak and higher overall level of per at the lower temperature. In contrast, there is no cycle in gl60j at either temperature, and levels of per are significantly different from WT and norpAP41 (Collins, 2004).

Therefore, to entrain locomotor behavior to different seasons, the fly's clock must respond to changes in both light and temperature. This is mediated through a molecular switch, whereby increases in temperature repress the splicing of an intron within the 3' UTR of per, delaying the onset of evening locomotor activity. Light also represses splicing, with higher splicing levels seen in shorter photoperiods, allowing locomotor activity to be fine-tuned to any given set of photoperiodic and temperature conditions. During the first day of DD, the level of splicing rises continuously. This is presumably because at the beginning of DD, the level of splicing is set low from the previous day's light input. Normally the light from the next day maintains this repressed level of splicing, but because this light input is absent, the repression of splicing is lifted, leading to a gradual rise in splicing levels (Collins, 2004).

The most obvious source for light input into the splicing machinery is the circadian photoreceptor Cry. However, analysis of the splicing levels in cryb shows that, although this mutation has an effect on splicing levels at 18°C, this effect is marginal and is seen only after lights off. This implies (1) that any function of Cry in the repression of splicing is not via the activation of this molecule by light; (2) because Cry is relatively dispensable for circadian locomotor rhythmicity per se, it also suggests that any minor role in splicing at low temperature is unrelated to the functioning of the clock. As the temperature rises, Per, Tim, and Cry all become involved in the regulation of per mRNA splicing. At 29°C, all three mutants show the same splicing phenotype, with ~30% of transcripts spliced during the day, but at night splicing is enhanced to ~45%. Although Per, Tim, and Cry are known to associate in light conditions, Cry and Tim can also associate in darkness, so it is not unexpected that the elimination of any one of the three proteins has a similar effect. Night time is also when the levels of these proteins are at their highest, and therefore any effects would be maximal (Collins, 2004).

At 29°C and in the presence of light, the levels of splicing in per01; cryb are elevated above those of either single mutant, which are themselves similar to WT. This suggests that the presence of either Per or Cry is required for light to repress splicing at 29°C. After lights off, the elevated levels of splicing of per are very similar in per01, tim01, cryb, and per01; cryb. Therefore Per, Tim, and Cry probably work together to repress splicing in the dark at 29°C. An alternative view for the virtually identical per01, tim01, and cryb splicing levels at 29°C is that this reflects a masking effect of light, so that exogenous LD cycles have a greater effect on splicing at night compared to WT, which shows a modest but significant day-night rhythm. Such stronger masking effects on locomotor behavior have also been observed in cryb mutants, but any mechanism that might relate or explain these observations remains obscure (Collins, 2004).

The examination of whole head homogenates means that the majority of biological material is derived from the eyes so may not represent exactly what occurs in the pacemaker neurons. The eyes are peripheral clocks, and the cryb mutation stops the cycling of the clock in whole head homogenates, although cycling continues in the pacemaker cells. One possibility is that the splicing observed in cryb does not truly reflect the role of Cry in setting splicing levels but is instead a consequence of the clock having stopped in the eyes, thus explaining why per01, tim01, and cryb all show the same splicing phenotype. However, if this splicing phenotype is simply what happens when the clock stops, then per01; cryb should show the same splicing phenotype as either single mutant. This is not the case, because the daytime splicing in per01; cryb at 29°C is dramatically elevated compared to either single mutant. Thus the splicing phenotypes of per01, tim01, and cryb cannot simply be a result of the clock having stopped. This means that it is the presence of these proteins, rather than their clock-dependent cycling, that is important to the regulation of per splicing levels (Collins, 2004).

In gl60j, there is no per mRNA cycle in whole head homogenates. This means that in the majority of cells in the gl60j head, the clock has either stopped or cells have become desynchronized. If the former is true, then splicing levels of gl60j should resemble those of per01 or tim01, and this is clearly not the case. If the latter is true, this could prevent the observation of any splicing rhythm, but the level of splicing observed should still represent the average level of splicing in this mutant background, which is clearly significantly different from WT. In any case, splicing levels observed in all visual mutants are likely to represent the effect of removing visual photoreception, because these elevated levels are similar to those observed in WT in DD (Collins, 2004).

norpAP41 and gl60j have considerably higher splicing levels than WT and cryb mutants at both temperatures, indicating that information received via the visual system rather than Cry drives this repression of splicing, which is borne out by analysis of gl60j cryb and norpAP41; cryb double mutants. The splicing levels of gl60j and gl60j cryb are similar at both temperatures, which is also true of norpA and norpAP41; cryb at 29°C. At 18°C, there is slightly more spliced per RNA in norpAP41; cryb than in the norpAP41 single mutant, reflecting the earlier result where cryb showed a marginal enhancement of splicing at cooler temperatures. These results also demonstrate that unspecific genetic background effects are not responsible for this marginal effect of cryb, because the double mutant background should make any interacting loci heterozygous. This lack of significant background effects in determining overall splicing levels has been confirmed by examining several natural European D. melanogaster lines. All mutants studied here show the same significantly enhanced splicing patterns when compared to any of the wild-caught isolates (Collins, 2004).

Unlike the clock and cryb mutants, there is no day-night difference in splicing levels at 29°C in either gl60j or norpAP41. One possibility is that visual system structures are required for the repression of splicing even in the dark, hence the overall elevated splicing levels in norpAP41 and gl60j at all times. This would be surprising, because such a role would obviously have to be light independent. More likely, the light input received through the eyes sets the splicing level during the day, and the clock maintains this repression at night. Thus, if the visual input is removed or reduced, as in DD, gl60j, or norpAP41 mutants, or in shorter photoperiods, then the subsequent splicing level is set higher. The difference in roles between cry and the visual system on per splicing levels may also partly explain recent observations that cryb mutants are able to adapt the timing of locomotor activity to long and short photoperiods, whereas flies with defective visual photoreception, including gl60j, are not (Collins, 2004).

Interestingly, although gl60j is the more severe visual mutant, norpAP41 has significantly higher per splicing levels than gl60j at both 18°C and 29°C. Additionally, whereas the difference between splicing levels at 18°C and 29°C is maintained in gl mutants (~65% and ~45% of transcripts spliced vs. ~45% and 25% in WT at 18°C and 29°C, respectively), this is greatly reduced in norpAP41 (65% and 55%). One possible explanation for this is that norpA may be a signaling molecule in the temperature-sensing pathway for the clock. The patterns of locomotor activity support a role for norpA in temperature sensing, with the norpAP41 fly's locomotor patterns seemingly more sensitive to high temperatures than WT. Additionally, norpAP41 evening locomotor activity peaks early at both 18°C and 29°C, and per mRNA splicing shows a corresponding elevation compared to WT. These are responses associated with low temperatures in WT D. melanogaster, and therefore norpAP41 mutants behave as if they have an impaired ability to detect high temperatures. norpAP41 flies still detect temperature changes (witness the altered evening peaks and splicing levels); they just react as if the temperature is colder than it actually is (Collins, 2004).

Thus, the enhanced per splicing seen in norpAP41 may reflect a direct link between norpA-encoded PLC signaling and the temperature sensitivity of the splicing mechanism, independent of norpA visual function. In the phototransduction cascade, rhodopsin activates a G-protein isoform that in turn activates the PLC encoded by norpA. As a result of this activation, Ca2+ permeable light-sensitive channels are opened, including members of the transient receptor potential (TRP) class. Recently it has been demonstrated that dANKTM1, a D. melanogaster TRP channel, is activated by temperatures from 24°C to 29°C. In addition, D. melanogaster painless mutant larvae have a disrupted TRP channel and display defective responses to thermal stimuli. Because several TRP family members act as thermal sensors in mammals, TRP channels appear to have an ancient heat-sensing function that is retained in both vertebrates and invertebrates. Given that this study has identified a heat-sensing role for norpA, and norpA is known to activate TRP channels in photoreception, it is not unreasonable to suppose that norpA plays a general role in responses to temperature stimuli (Collins, 2004).

per splicing levels may also impact on aspects of behavior other than the timing of evening locomotor activity. For instance, the free-running period of norpAP41 is ~1 h shorter than WT. The splicing levels of per mRNA are greatly elevated in this background, and elevated splicing is predicted to advance the Per protein cycle and thus speed up the clock. In fact, the splicing mechanism should have the effect of speeding up the clock at colder temperatures and slowing it down at high temperatures, thereby providing a potential basis for temperature compensation (Collins, 2004).

The position of the evening activity peak at different temperatures moves in different mutant backgrounds. For WT, norpAP41, and cryb, the level of splicing appears to correlate with the position of the evening activity peak at different temperatures. At 18°C, there is a small but significantly greater relative amount of spliced per RNA in cryb than in WT, resulting in the earlier evening activity peak seen in cryb flies. This difference in per splicing is greatest after lights off at both temperatures. This is when Per levels will be rising, because Tim is present for Per stabilization, so enhancement of Per accumulation by elevated per splicing is likely to have its most noticeable effect around dusk or early evening. A similarly consistent situation is seen in norpAP41: there is more spliced per mRNA present at 18°C (65%) than 29°C (55%), accounting for the earlier peak of evening activity at 18°C. Additionally these levels are higher than those seen in either WT (45% and 25% per transcripts spliced at each temperature) or cryb (55% and 40%) and relates to the earlier phases of locomotor activity seen in norpAP41 compared to the other genotypes. However, at 18°C there is more spliced per in norpAP41 than in cryb, but the evening activity peak occurs at the same time. The simplest explanation is that there is a limit to how early the evening activity peak can occur, no matter what the per splicing level, because splicing alters the accumulation of Per protein; this is limited by the light-dependent degradation of Tim. Therefore, in general, the level of splicing determines when the peak level of locomotor activity will occur (Collins, 2004).

The level of splicing of the per intron cannot be the only determinant of evening peak position, because the relationship between the per splicing level and evening activity peak position breaks down in norpAP41; cryb and gl60j, where there are different levels of splicing at the two temperatures but no corresponding difference in the evening peak position. When the underlying per mRNA cycles of gl60j, norpAP41, and WT flies were analyzed at 18°C and 29°C, it was found that whereas per levels cycle in norpAp41 and WT, this cycle is lost in gl60j. If there is no underlying per RNA cycle, then there is no mRNA peak to be advanced or delayed by splicing (Collins, 2004).

At the cellular level, although gl is not a clock component, when mutated, it eliminates a number of clock-expressing cells within the head, including the eyes, ocelli, Hofbauer-Buchner (H-B) eyelet, and the dorsal neuron 1 (DN1) cells. Despite this, the primary effect on the clock is to remove most of the visual entrainment pathway, but the clock in the key pacemaker cells of gl60j mutants must still be functional, because behavior still entrains to LD cycles and remains rhythmic in DD. It is significant that the crosstalk between different classes of clock cells is essential for the generation of robust behavioral rhythms. Thus loss of the overall per mRNA rhythm may be a consequence of disrupting this network in gl60j, and, while leaving the basic system intact, this affects the more subtle temperature-sensitive aspects of entrainment. A similar argument based on an interruption of the entrainment network can also be proposed to explain the corresponding results with norpAP41; cryb double mutants, because in this case per mRNA is assumed to be noncycling because of the cryb background. However, the locomotor behavior of cryb single mutants remains thermosensitive even though overall per mRNA is noncycling. Thus, only when the photoreceptive pathway and mRNA cycle are both compromised (as in gl and norpAP41; cryb) is locomotor behavior insensitive to temperature-dependent changes in per splicing levels (Collins, 2004).

A model is presented of how light and temperature may set the splicing level of the clock. How temperature is detected by the splicing machinery is not yet clear, but there is compelling evidence that norpA plays a role. At low temperatures, the splicing level is primarily set by light via the visual system rather than Cry, which is then remembered during the night. In longer periods of darkness such as in DD, this memory decays, and splicing levels begin to rise. Thus the visual system represses splicing by enhancing the effects of an unknown repressor molecule(s) that is sensitive to temperature change and the norpA PLC. At high temperatures, the regulation of splicing is more stringent and complex and recruits the circadian clock. Again, the light input received through the visual system sets the low splicing level during the day. This appears to also depend on the presence of at least two of the three molecules, Per, Tim, or Cry, because elimination of any one of these gives a barely detectable daytime rise in splicing, reflecting the very low levels of Per, Tim, and Cry at this time. However, elimination of both Per and Cry in the per01; cryb double mutant lifts all light-dependent repression during the day (Collins, 2004).

At night, the level of splicing set during the day by the visual system is again remembered and maintained by the clock at night. If per, tim, or cry is eliminated, then this repression of splicing is lost at night, generating the day/night difference in splicing levels. In gl60j cryb or norpAP41; cryb, because there is no visual light input during the day, there is no splicing level for the clock to remember, and therefore there is no day/night difference in splicing levels. Thus at high temperature, the visual system activates the repressor molecule during the day, and the clock maintains this activation at night. It is assumed that recruiting the clock at high temperature to inhibit per splicing is required to ensure that the fly's locomotor/foraging behavior is adaptive and does not encroach on those times of the day when there would be a significant risk of desiccation (Collins, 2004).

Disruption of Cryptochrome partially restores circadian rhythmicity to the arrhythmic period mutant of Drosophila

The Drosophila circadian clock is generated by interlocked feedback loops, and null mutations in core genes such as period and timeless generate behavioral arrhythmicity in constant darkness. In light-dark cycles, the elevation in locomotor activity that usually anticipates the light on or off signals is severely compromised in these mutants. Light transduction pathways mediated by the rhodopsins and the dedicated circadian blue light photoreceptor cryptochrome are also critical in providing the circadian clock with entraining light signals from the environment. The cryb mutation reduces the light sensitivity of the fly's clock, yet locomotor activity rhythms in constant darkness or light-dark cycles are relatively normal, because the rhodopsins compensate for the lack of cryptochrome function. Remarkably, when a period-null mutation was combined with cryb, circadian rhythmicity in locomotor behavior in light-dark cycles was restored, as measured by a number of different criteria. This effect was significantly reduced in timeless-null mutant backgrounds. Circadian rhythmicity in constant darkness was not restored, and Tim protein did not exhibit oscillations in level or localize to the nuclei of brain neurons known to be essential for circadian locomotor activity. Therefore, this study uncovered residual rhythmicity in the absence of period gene function that may be mediated by a previously undescribed period-independent role for timeless in the Drosophila circadian pacemaker. Although a molecular correlate for these apparently iconoclastic observations is not available, a systems explanation for these results is provided, based on differential sensitivities of subsets of circadian pacemaker neurons to light (Collins, 2005).

This study has revealed a surprising and intriguing restoration of circadian rhythmicity in LD cycles in per01; cryb flies. This partial rescue can even be extended to the adaptive thermal change in locomotor behavior mediated by 3' UTR splicing of the per transcript (Collins, 2004: Majercak, 2004; Majercak, 1997). A number of criteria have been used to dissect rhythmic behavior, including phase shifting in response to light pulses in LD and the use of T cycles to suggest that a residual oscillation, rather than an hourglass, underlies the behavior of the double mutant. The phase shifting of the per01; cryb oscillator is particularly informative because per01 is effectively rescuing this phenotype in cryb. This can be understood in terms of the robust, high-amplitude oscillator in cryb, being less 'perturbable' by light as Cry photoreception is lost, whereas the damped oscillator in per01; cryb is more sensitive to the environmental stimulus, precisely because of its low amplitude. The damped oscillation in the per01; cryb double mutant can be eliminated by removing tim function, but this is temperature dependent, so tim cannot supply the full explanation for these residual cycles. Although these experiments have focused on the 'evening' oscillator, of related interest is that the residual 'morning' oscillator that anticipates the lights-on signal in per01 was also observed. It is clear that both of these studies raise again the possibility of an underlying rhythmicity in per01 flies that was initially suggested from statistical analyses of mutant locomotor records (Collins, 2005).

The entrainment of a frequency-less oscillator in Neurospora crassa has been the subject of some recent debate, and the parallels with a residual rhythmicity in per-null Drosophila are striking. Furthermore, the rescue of per01 behavior by cryb would appear, at least superficially, to be similar to the situation in mammals in which a Cry mutation restores free-running rhythms to the arrhythmic mPer2 mutant mouse; this has been explained in terms of the freeing up in the double mutant of other mPer and Cry paralogues to interact and restore the original behavior. Since the fly does not have paralogues of per and cry, an explanation must be sought elsewhere. The only other genotypes identified so far with an anticipatory locomotor activity peak in LD and loss of rhythmicity in DD are disconnected (disco) and Pdf0. Neither mutation affects the molecular core of the circadian clock, rather the network of pacemaker neurons is disrupted. PDF is required for the functional integration of several clock neuronal groups within the brain, suggesting that disruption of interneuronal signaling causes arrhythmic behavioral output in the absence of synchronizing cues. In arrhythmic disco mutants, the clock gene expressing lateral neurons (LNvs and LNds) are usually absent, whereas the dorsal neurons are still present, thus indicating that the former are necessary for self-sustained rhythmicity, whereas the latter can only mediate rhythmic behavior under LD conditions (Collins, 2005).

This networking of clock neurons provides a basis for possible models to explain LD behavioral anticipation in the absence of Per, based on functional differences between the three groups of clock genes expressing LNs. Of these, only the small ventral LNs (sLNvs) and dorsal LNs (LNds) have a self-sustaining molecular clock when initially released into DD, although the latter depends on the former for synchronization. The third group, the large ventral LNs (l-LNvs) do not have a self-sustaining clock, although after a few days, tim mRNA again begins to accumulate rhythmically in these cells. Furthermore, rhythmic Tim expression is more sensitive to disruption by cry mutations in the l-LNvs, than in the s-LNvs or the LNds under LD conditions, suggesting that rhythmic output from the l-LNvs are compromised in a cryb background. In turn, this may contribute to the peculiar defects of cryb that includes robust entrainment to LD cycles, but significantly reduces behavioral phase shifts to brief light pulses, and, unlike wild-type, the maintenance of rhythmic behavior in constant light (Collins, 2005).

In the favored model, the robust s-LNv and LNd oscillators in cryb 'resist' the effects of brief light pulses, because of the impaired light input that is relayed to the s-LNvs, and from the s-LNvs to the LNds, by the more light-relevant l-LNvs. In per01, the molecular clock is severely dampened in all clock neurons, more so in the s-LNvs and LNds that have an endogenous cycle than the l-LNvs that do not. Thus, the light-mediated input from the l-LNv neurons into the s-LNvs, and indirectly to the LNds, is no longer resisted, and now overwhelms the residual damped per01 oscillator in these neurons, stimulating light-induced non-rhythmic locomotor behavioral output. However, when cryb and per01 are combined, the weak oscillator of per01 is no longer overcome by the light input because it is attenuated by cryb and mediated via the l-LNvs. Thus, rhythmic behavior is observed in LD cycles, providing a glimpse of the residual Per-independent, partly Tim-regulated clock. This model is preferred over a simpler one in which only the s-LNvs are involved, because previous studies have shown that the only direct photoreceptive input into these neurons is from the Hofbauer-Buchner eyelet, which is a very weak photoreceptor at best and it cannot, in the absence of other photoreceptors, entrain the fly's behavior (Helfrich-Forster, 2002). Thus, it is difficult to see how light information would be received by the s-LNvs to entrain the per01; cryb double mutant so effectively, unless it is transmitted from another neuronal source: the l-LNvs (Collins, 2005 and references therein).

In support of the model, there appears to be both direct and indirect neural connections between the compound eyes and the l-LNvs, suggesting that the l-LNvs may act as the light 'amplifier'. This study extends earlier observations by showing that photoreceptor cells expressing the rhodopsin genes, Rh3 and Rh5, send their axons through the medulla terminating in close proximity to the general region where the l-LNvs likely extend their dendritic arborizations..Although not definitive, these results support earlier claims that the photoreceptors may directly (or indirectly) synapse with the l-LNvs. As stated above, these molecular and proposed functional differences between s- and l-LNvs may also contribute to explaining the loss of light responsiveness in cryb mutant flies, which are blind to constant light and brief light pulses, despite retaining light input from the canonical visual transduction pathway. Thus Cryptochrome, aside from being a photoreceptor in its own right, also appears to control a gateway for rhodopsin-mediated light input into the clock (Collins, 2005).

Although the disruption of neural networks in this way probably explains the light responses of the clock in per01; cryb, it offers no molecular basis for the observed behavior. The loss of anticipation in tim-null-bearing genotypes suggests that Tim may play a key role. Although no significant nuclear Tim was observed in the LNvs or LNds of per01; cryb, the latter neurons being particularly relevant for providing the evening peak of locomotor activity present in the double mutants, it is suspected that Tim is shuttling continually in and out of the nucleus because Tim can enter the nucleus alone, but requires Per for nuclear retention, at least in larval clock neurons. Once in the nucleus, Tim is presumably interacting with as yet unidentified protein(s) in a light-dependent manner, generating behavioral rhythms in the double mutants. A microarray study found that 18 of the 72 genes that cycled in LD in wild-type also cycle in per01. Any one or more of these light-controlled proteins could therefore interact with Tim, contributing to the light-dependent oscillator of per01; cryb. In fact, it has been noted by others that a glutamine-rich transcriptional activator domain found within Tim may allow it to regulate other genes in a Per-independent manner (Collins, 2005).

Veela defines a molecular link between Cryptochrome and Timeless in the light-input pathway to Drosophila's circadian clock

Organisms use the daily cycles of light and darkness to synchronize their internal circadian clocks with the environment. Because they optimize physiological processes and behavior, properly synchronized circadian clocks are thought to be important for the overall fitness. In Drosophila, the circadian clock is synchronized with the natural environment by light-dependent degradation of the clock protein Timeless, mediated by the blue-light photoreceptor Cryptochrome (Cry). This paper report identification of a genetic variant, Veela, which severely disrupts this process, because these genetically altered flies maintain behavioral and molecular rhythmicity under constant-light conditions that usually stop the clock. The Veela strain carries a natural timeless allele (ls-tim), which encodes a less-light-sensitive form of Timeless in combination with a mutant variant of the F-box protein Jetlag. However, neither the ls-tim nor the jetlag genetic variant alone is sufficient to disrupt light input into the central pacemaker. A strong interaction between Veela and cryptochrome genetic variants, demonstrating that the Jetlag, Timeless, and Cry proteins function in the same pathway. Veela also reveals a function for the two natural variants of timeless, which differ in their sensitivity to light. In combination with the complex array of retinal and extraretinal photoreceptors known to signal light to the pacemaker, this previously undescribed molecular component of photic sensitivity mediated by the two Timeless proteins reveals that an unexpectedly rich complexity underlies modulation of this process (Peschel, 2006).

Veela is abnormally rhythmic in constant light, similar to mutations affecting the blue-light photoreceptor Cry. Veela's phenotype is due to the simultaneous presence of the ls-tim allele (encoding a less-sensitive form of Tim) and the jetc variant encoding a mutant form of the F-box protein Jet. Veela genetically and molecularly interacts with cryb, indicating that Tim, Jet, and Cry function in the same circadian light-synchronization pathway. These findings show that additional factors are necessary to elicit the phenotypes previously associated with jet variants. In particular, only when jetc is linked to the ls-tim allele, which encodes a less-light-sensitive form of Tim, can abnormal behavioral rhythmicity in LL be observed. The importance of the Jet protein per se in the light-entrainment process remains unclear, also when considering certain aspects of the original jet study in conjunction with the findings presented in his study. All control flies used by Koh (2006) came from a y w genetic background, which carries the s-tim allele. Contrarily, all jetc or jetr mutant flies carried the ls-tim allele (necessarily; otherwise, they would have behaved like WT). It follows that behavioral and molecular differences between control and mutant flies reported by Koh in fact reflect the combined effects of ls-tim (vs. s-tim) and jetc (vs. jet +). In conjunction with Western blot data showing an increased jet-independent stability of the larger Tim form compared with the smaller one, it seems that the effects on Tim degradation previously attributed to jet variants are mainly a reflection of the different features of the two Tim proteins. This may also explain why Koh saw only very subtle effects of their mutant Jet proteins on Tim degradation in vitro (Peschel, 2006).

Nevertheless, it is clear that jet influences the light-input pathway of the circadian clock; WT flies behave arrhythmically in LL, even though they carry ls-tim. Moreover, Veela strongly interacts with Cry, a crucial protein for circadian light input in flies. Importantly, these findings reveal that, with the current knowledge, an in vivo function for jet's F-box protein can be demonstrated only when the available jet variants are combined with ls-tim. To ultimately resolve the specific function of the Jet protein in the light-input pathway, loss-of-function jet mutants (Debruyne, 2006) or specific RNAi transgenics need to be generated and analyzed chronobiologically (Peschel, 2006).

Characterization of Veela also led to the assignment of a biological function for the two natural tim variants that were identified many years ago. This study has show that Tim encoded by the ls-tim allele is more stable after light exposure, and that this increased stability has behavioral consequences when flies are exposed to constant light; if the ls-tim allele is linked to jetc, these flies behave abnormally rhythmically in LL. If jetc is linked to s-tim, the flies behave like WT and become arrhythmic in LL. Therefore, the less-light-sensitive Tim form encoded by ls-tim is necessary and sufficient to block light input into the circadian clock of jetc flies. In nature, the natural polymorphism at the tim (and perhaps jet) locus might be used to fine-tune the light sensitivity of Drosophila's circadian clock on a purely molecular level. In conjunction with various anatomical light-input routes that are known to send light to Drosophila's circadian pacemaker, these findings reveal a glimpse of the potential complexity of this process. The frequent and random occurrence of tim and jet variants in currently used laboratory strains also speaks to a more cautious strain selection and genotyping in all studies concerning light-input pathways to the circadian clock (Peschel, 2006).

Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest-activity rhythms and relies upon different groups of PERIOD (Per)-expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional Cryptochrome (Cry), as opposed to wild-type flies, are rhythmic in constant light, transgenic flies were analyzed expressing Per or Cry in the LN-MO or LN-EO. Under constant light conditions and reduced Cry function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, this study shows that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. It is therefore proposed that day-night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons (Picot, 2007).

The PDF-expressing LNs and the PDF-negative LNs were previously characterized as morning and evening cells, respectively, in LD conditions. Furthermore, the morning LNs were able to drive robust 24-h rhythms in DD, whereas evening LNs were not. This study shows that in LL, the evening LNs drive robust rhythms when cryptochrome signaling is absent or reduced, whereas the morning cells are not able to do so. Surprisingly, the molecular oscillations of both groups can be uncoupled from behavioral rhythmicity, depending on light conditions. In DD, the two LN groups show autonomous molecular cycling, but there is no behavioral output when the LN-EO is cycling alone. In LL (and reduced Cry signaling), both groups still show autonomous cycling, but there is no behavioral output when the LN-MO is cycling alone. It is therefore concluded that light has opposite effects on the behavioral output of the two LN oscillators, activating it from the evening LNs and inhibiting it from the morning LNs (Picot, 2007).

The opposite effects of light on the behavioral outputs do not appear to be related to entrainment, since Per oscillations in both the PDF-positive and PDF-negative LNs are synchronized to the LD cycles even in the absence of Cry signaling. The inhibiting effect of light on the LN-MO behavioral output is abolished when the visual system is genetically ablated. This suggests that the projections of the visual system photoreceptors convey, not only input information to the PDF cells (light entrainment), but also signals to control their behavioral output (light inhibition). It is tempting to speculate that light exerts both effects through a direct connection of the PDF cells with the visual system. The Hofbauer-Büchner eyelet photoreceptors that project directly to the LN-MO neurons and participate in the entrainment provide a possible pathway (Picot, 2007).

It was recently reported that the overexpression of Per or of the Shaggy (Sgg) kinase in the PDF-negative clock neurons induced rhythmic behavior in LL. The rhythmicity was associated with the cycling of Per subcellular localization in some of the DNs, whereas the PDF-expressing cells were molecularly arrhythmic. These studies therefore concluded that some DN subsets are able to drive behavioral rhythms in LL. Different groups of PDF-negative cells may thus be able to drive behavioral rhythms in constant light, depending on whether and how the molecular clock is manipulated. Such manipulation could also directly affect molecular oscillations, making them less easy to detect. Since Cry does not appear to have a core clock function in the brain, these data are largely based on situations in which the clock mechanism is little if at all altered. The data support a major contribution of the LN-EO to the robust rhythms of cryb mutants in LL (Picot, 2007).

The strong rhythmicity of the cryb pdf0 double mutants in LL contrasts with their weak rhythmic behavior in DD. Altogether, these results strongly suggest that this robust rhythm is generated by the LN-EO, which would therefore behave as a PDF-independent autonomous oscillator. However, the period of the oscillator is clearly influenced by PDF signaling, and thus by the LN-MO, going from 24–25 h in cryb to 22–23 h in cryb pdf0 flies. An attractive possibility is that the strong short-period rhythm observed in the cryb pdf0 double mutant in LL has the same neuronal origin as the weak short-period rhythm described for pdf0 mutants in DD. The cellular basis of this PDF-independent oscillator in DD remains unclear, although the presence of similar rhythms in flies genetically ablated for the PDF-expressing neurons suggests that it originates from other clock cells (Picot, 2007).

Different results were obtained for the recently described DN-based LL oscillators. When transferred to a pdf0 background, all SGG-overexpressing flies were found to be arrhythmic, whereas about 60% of the Per-overexpressing flies displayed long-period rhythms. This suggests that different types of DNs with different sensitivity to PDF may have been analyzed in these two studies. Although some DNs may contribute to the PDF-independent rhythms, these data suggest a strong contribution of PDF-negative LNs to the rhythmic behavior that persists in pdf0 mutants. The weakness of the short-period rhythm of pdf0 flies in DD may reflect the inhibition of the LN-EO output in the absence of light (Picot, 2007).

These results indicate that whereas the LN-MO autonomously drives rhythmic behavior in constant darkness, the LN-EO plays this role in constant light, if Cry signaling is abolished or reduced. It is thus suggested that in natural LD conditions, Drosophila behavior could be driven by the LN-MO during the night, and by the LN-EO during the day, when cryptochrome is quickly degraded by light. This supports a model of a light-induced switch between the circadian oscillators of the LNs that would allow a better separation of the dawn and dusk activity peaks in day–night conditions. It has been shown that PDF-expressing LNs drive the clock neuronal network in short days, whereas PDF-negative DN subsets take the lead in long days. Thse results suggest that the PDF-negative cells of the LN-EO could also be a major player during the long days. Surprisingly, it was found that light does not seem to act on the molecular oscillations, but inhibits the LN-MO behavioral output and promotes the LN-EO behavioral output, which may provide an efficient fine tuning of the contributions of the two oscillators. It therefore appears that the visual system controls both the input (entrainment) and the behavioral output of the LN oscillators in the Drosophila brain clock. In species such the honeybee or the flour beetle, which appear to lack a light-sensitive Cry protein, this role of the visual system may be particularly important (Picot, 2007).

Identifying specific light inputs for each subgroup of brain clock neurons in Drosophila larvae

In Drosophila, opsin visual photopigments as well as blue-light-sensitive cryptochrome (Cry) contribute to the synchronization of circadian clocks. This study focused on the relatively simple larval brain, with nine clock neurons per hemisphere: five lateral neurons (LNs), four of which express the pigment-dispersing factor (PDF) neuropeptide, and two pairs of dorsal neurons (DN1s and DN2s). Cry is present only in the PDF-expressing LNs and the DN1s. The larval visual organ expresses only two rhodopsins (RH5 and RH6) and projects onto the LNs. PDF signaling is required for light to synchronize the Cry- larval DN2s. This study shows that, in the absence of functional Cry, synchronization of the DN1s also requires PDF, suggesting that these neurons have no direct connection with the visual system. In contrast, the fifth (PDF-) LN does not require the PDF-expressing cells to receive visual system inputs. All clock neurons are light-entrained by light-dark cycles in the rh52;cryb, rh61 cryb, and rh52;rh61 double mutants, whereas the triple mutant is circadianly blind. Thus, any one of the three photosensitive molecules is sufficient, and there is no other light input for the larval clock. Finally, it was shown that constant activation of the visual system can suppress molecular oscillations in the four PDF-expressing LNs, whereas, in the adult, this effect of constant light requires Cry. A surprising diversity and specificity of light input combinations thus exists even for this simple clock network (Klarsfeld, 2011).

The larval brain clock and its light inputs are generally considered much simpler than their adult counterparts. We find here that larvae, with only nine clock neurons and 12 photoreceptors on each side, nevertheless display four distinct combinations of light inputs (Klarsfeld, 2011).

Anatomical data and the present work show that PDF+ LNs are the only brain cells to perceive light both cell autonomously (via CRY) and through a direct connection to the visual system. They thus appear to be the main players responsible for synchronizing the larval brain clock network to LD cycles. The DN2s, in contrast, possess neither type of light input, but play a major role in the temperature entrainment of the clock (Picot, 2009). Previous studies have shown that the DN2s are intrinsically blind and must rely on PDF signaling from the LNs to synchronize to LD cycles (Picot, 2009). This study shows that the other dorsal group, the DN1s, is also sensitive to PDF signaling. In the absence of functional Cry, Pdf is required to synchronize DN1s by light, as demonstrated by the lack of Per oscillations in the DN1s of the cryb pdf0 double mutant. This is consistent with the presence of a dendritic-like arborization from the DN1s close to the dorsal projection of the LNs. On the other hand, it tends to exclude a functional connection between the DN1s and the larval visual system, in agreement with the absence of DN1 neurites reaching the Bolwig's nerve terminals (Klarsfeld, 2011).

The Pdf-dependent entrainment of both DN1s and DN2s by the visual system also indicates that the fifth LN, although projecting largely like the Pdf+ LNs, cannot synchronize the DNs. However, the fifth LN might be involved in RH5-dependent acute larval responses to light, which do not require the Pdf+ LNs. The entrainment of the fifth LN in the absence of both Cry and the Pdf+ LNs suggests a direct connection to the visual system, in agreement with its arborization in the larval optic neuropil. Recent single-cell analysis indeed revealed this arborization to be even broader than that of the Pdf+ LNs. However, such connection to the visual system does not allow constant light to disrupt Per oscillations in the fifth LN, contrary to the Pdf+ LNs, suggesting different downstream signaling in these two types of visual system targets. Finally, the results suggest a hitherto unsuspected connection between some Cry+ neurons and the fifth LN. This connection does not rely on Pdf and could be directly from the DN1s or the Pdf+ LNs, which both have projections in the vicinity of the fifth LN's projections (Klarsfeld, 2011).

More generally, the fact that the Cry- fifth LN and DN2s display normal Per oscillations in the absence of a functional visual system is consistent with Cry transmitting light information in a non-cell-autonomous way. This has already been proposed in the adult brain for the three Cry- dorsal lateral neurons and the DN2s. However, it remains possible that such nominally Cry- cells in the adult express very low levels of Cry, as judged from reporter gene expression. In contrast, Cry expression in the larval 5th LN and DN2s was observed neither with antibodies, not with any reporter lines. The present results make it even less likely, because constant light does not affect these neurons at all (Klarsfeld, 2011).

The role of the Pdf neuropeptide in the light entrainment of the DN1s and DN2s appears somewhat different for the two subgroups. First, Pdf sets the DN1s and DN2s to very different phases: the DN2s are set in antiphase with the LNs, whereas the DN1s are set in phase with the LNs. This suggests that the corresponding signaling cascades differ somewhere downstream from the Pdf receptor. In addition, the dispersion of cell labeling intensities suggests that unentrained DN2s oscillate, although asynchronously (even within a single brain hemisphere), while unentrained DN1s do not, but rather express constant, moderate Per and TIM levels. The same may hold true for completely blind larvae. While the LNs and DN2s seem to oscillate with random individual phases, all DN1s display very similar Per levels. This implies that, in LD, Pdf may be needed not only to synchronize but to trigger (or at least maintain until the third larval stage) DN1 oscillations in the absence of Cry activation. In contrast, Pdf synchronizes persistent autonomous oscillations in the DN2s. The non-autonomous cycling of the Cry-expressing DN1s suggests that they may have an important role in synchronizing the network to LD cycles. Conversely, the capacity of the DN2s for autonomous cycling in the absence of light cues may relate to their specific role in temperature entrainment (Klarsfeld, 2011).

Lack of entrainment by light was previously reported for the LNs and the DN1s in norpAP41;;cryb larvae, while, rather surprisingly, molecular oscillations were still detected in their DN2s. While the DN2s require Pdf to entrain in LD, they appear to entrain to temperature cycles very efficiently on their own (Picot, 2009). This means one cannot exclude the possibility that, in a previous study, small temperature changes induced by the LD cycles weakly entrained these neurons, but not the others. Alternatively, the DN2s might collect light information from a NORPA-independent pathway. NORPA-independent photoreception appears to participate in adult circadian photoreception (Klarsfeld, 2011).

The results show that RH5, RH6, and Cry are the only light input pathways for synchronizing the larval clock neurons to LD cycles. RH5, RH6, and Cry are each sufficient alone to entrain all these neurons, whereas, in the adult, some clock neurons fail to entrain in the absence of Cry. At least two more rhodopsins, including RH1 and a UV-blue one (RH3 and/or RH4), participate in the adult, so that all available rhodopsins in the adult eye may also be involved in entraining the clock. Recently, at least two classes of larval sensory neurons, outside BO, have been shown to express visual transduction components. One of these two is involved in thermal preferences, with RH1 as the presumed temperature sensor, while the other mediates rhodopsin-independent avoidance of very high light intensities. At least in the conditions used in this study, these novel sensory pathways do not seem to contribute to circadian light entrainment (Klarsfeld, 2011).

Interestingly, constant light, acting Cry independently through the visual system, can abolish or greatly disturb oscillations in the Pdf+ LNs of larvae but not adults. Similarly, the larval visual system is required for fast TIM degradation in the LNs at the end of the night. The Pdf+ LNs of eyeless adult flies, in contrast, seem to respond normally even to a very short light pulse, suggesting that the visual system is dispensable for the response to light pulses in adults, but not larvae. The different sensitivity of the larval clock to visual system inputs could be related to the change in signaling pathways that occurs as the larval cholinergic visual system develops into the adult histaminergic visual system. Moreover, contrary to the adult situation, the larval visual nerve may be light sensitive all along its length, down to its connection with the LNs, as judged from RH6 and NORPA expression. How visual system signaling ultimately affects the clock, whether in larvae or adults, remains to be discovered (Klarsfeld, 2011).

Both RH5+ and RH6+ BO photoreceptors contribute to the light responses of the larval brain clock that were tested, i.e., entrainment in LD and disruption of LN rythmicity in LL. Similarly, both photoreceptor types are equally able to suppress TIM levels in the LNs after a 2 h light exposure at the beginning of the night. In contrast, RH5 fibers alone specifically mediate acute larval responses to light, while RH6 fibers alone are specifically required for the development of a serotonergic arborization that also contacts the LNs. That RH6 activation strongly disrupts molecular oscillations in the LNs even in RR was, however, not anticipated. In the adult, RR does not affect molecular or activity rhythms (Klarsfeld, 2011).

Activation of RH6 above 600 nm is less than a few percent of peak activation at ~510 nm. This suggests that the clock of the larval LN is extremely sensitive to red light, which may explain why no larval activity rhythm was recorded in a study that used video tracking in constant red light. A strong sensitivity of larvae to the more penetrating, longer wavelengths of light may be related to their burrowing lifestyle (Klarsfeld, 2011).

Dopamine acts through Cryptochrome to promote acute arousal in Drosophila

The fruit fly, Drosophila melanogaster, is generally diurnal, but a few mutant strains, such as the circadian clock mutant ClkJrk, have been described as nocturnal. This study reports that increased nighttime activity of Clk mutants is mediated by high levels of the circadian photoreceptor Cryptochrome (Cry) in large ventral lateral neurons (l-LNvs). Cry expression is also required for nighttime activity in mutants that have high dopamine signaling. In fact, dopamine signaling is elevated in ClkJrk mutants and acts through Cry to promote the nocturnal activity of this mutant. Notably, dopamine and Cry are required for acute arousal upon sensory stimulation. Because dopamine signaling and Cry levels are typically high at night, this may explain why a chronic increase in levels of these molecules produces sustained nighttime activity. It is proposed that Cry has a distinct role in acute responses to sensory stimuli: (1) circadian responses to light, as previously reported, and (2) noncircadian effects on arousal, as shown in this study (Kumar, 2012).

Both dopamine and Cry are required for acute arousal at night. An arousal-promoting role for dopamine is supported by earlier studies. Dopamine transporter mutants were shown to exhibit a decreased arousal threshold, whereas the pale mutants exhibit an increased arousal threshold. This effect on arousal reflects a novel role for dopamine in sensory responses at night. Cry has not been implicated in arousal, although it promotes neural activity in a light-dependent manner. As in the case of the neural activity assay, this study found that arousal in response to sensory stimuli is reduced but not eliminated by the cryb mutant, indicating that the mechanism is distinct from the circadian response that is eliminated by cryb. Both neural activity and behavioral arousal responses are eliminated by the cry0 mutant, suggesting that the neural response underlies the behavioral effect. It is proposed that Cry is required at multiple levels for acute responses to sensory stimuli. In the case of circadian photoreception, it is absolutely required for phase-shifting in response to pulses of light, although not for entrainment to LD cycles. In the case of responses to sensory stimuli, again it is required for the startle response. Any effects of Cry on light-induced activity (physiological or behavioral) are likely to be acute, since Cry gets degraded with increased light treatment. Interestingly, in two different species of Bactrocera, cry mRNA levels are positively correlated with the timing of mating, which is also indicative of a regulated response required for a specific purpose. A chronic effect is seen only in the case of Drosophila Clk mutants, where levels of Cry are considerably higher than normal, and dopamine signaling is also elevated. It is hypothesized that Cry only promotes nocturnal activity in flies with chronically elevated dopamine signaling because dopamine acts as a trigger to activate Cry. However, this activation may be different from activation in a circadian context, given that different mechanisms appear to underlie the circadian and arousal-promoting roles of Cry. Dopamine- and Cry-mediated locomotor activity is restricted largely to the night because of light-induced Cry degradation and light-induced inhibition of dopamine signaling (Kumar, 2012).

At night, animals sleep, and the arousal threshold is increased. However, they still need to be able to respond in case of sudden events. It is speculated that dopamine and Cry are essential for this. In the case of Cry, it may arouse the animal and also reset the clock. For instance, the immediate response of an animal to a pulse of light at night is to wake up, which may be driven by the arousal-promoting role of Cry. In addition, the circadian clock must be reset, which requires the circadian function of Cry. Whether or not these roles of Cry are conserved, it is speculated that dopamine functions similarly in mammals. Interestingly, melanopsin, which is the circadian photoreceptor in mammals (analogous to Cry in flies), is regulated by dopamine in intrinsically photosensitive retinal ganglion cells (ipRGCs). Like Cry, melanopsin is also required for acute behavioral responses to light, specifically for sleep induction in nocturnal animals during the day. These ipRGCs have been proposed as functionally similar to l-LNvs, so a conserved function for the relevant molecules is intriguing. Finally, it is noted that elevated dopamine has been linked to increased nighttime activity in humans, which are, of course, diurnal like Drosophila. People with Sundown syndrome or nocturnal delirium show increased agitation and sleep disturbances in the early evening, which can be treated with anti-psychotic medications that target dopamine signaling (Kumar, 2012).

Phase-shifting the fruit fly clock without cryptochrome

The blue light photopigment cryptochrome (CRY) is thought to be the main circadian photoreceptor of Drosophila melanogaster. Nevertheless, entrainment to light-dark cycles is possible without functional CRY. This study monitored phase response curves of cry01 mutants and control flies to 1-hour 1000-lux light pulses. It was found that cry01 mutants phase-shift their activity rhythm in the subjective early morning and late evening, although with reduced magnitude. This phase-shifting capability is sufficient for the slowed entrainment of the mutants, indicating that the eyes contribute to the clock's light sensitivity around dawn and dusk. With longer light pulses (3 hours and 6 hours), wild-type flies show greatly enhanced magnitude of phase shift, but CRY-less flies seem impaired in the ability to integrate duration of the light pulse in a wild-type manner: Only 6-hour light pulses at circadian time 21 hours significantly increased the magnitude of phase advances in cry01 mutants. At circadian time 15 hours, the mutants exhibited phase advances instead of the expected delays (Kistenpfennig, 2012).

There is one main difference between wild-type and CRY-deficient flies regarding parametric light effects: cry mutants do not become arrhythmic at LL, not even at high irradiances. In this respect, the clock of CRY-deficient flies appears similar to that of mammals because the clock of most mammalian species runs under constant dim light. On the molecular level, this difference is easy to understand because light-activated Drosophila CRY leads to degradation of TIM. After TIM has disappeared, PER cannot be stabilized, and as a consequence, the clock stops. Indeed, it has been noted that after 6-hour light pulses, the activity rhythm of wild-type flies always started with the same phase, suggesting that the clock had completely stopped and was restarted after lights-off. Mammalian-like CRY is not light sensitive, and thus, light will probably not completely stop the mammalian clock, at least not after light pulses of 6 hours. Only a longer light exposure will stop the clock, as recently reported in mice after a pulse longer than 15 hours (Kistenpfennig, 2012).

The phase response curve for 12-hour light pulses shows that the clock of CRY-less flies is mainly light responsive at dawn and dusk. Such temporally restricted sensitivity must be sufficient for entrainment because dawn and dusk are the most important times at which a clock needs to respond to light. Because the light sensitivity of CRY-less flies is mediated by photoreceptor organs (as the compound eyes, the H-B eyelets, and possibly the ocelli), the current results suggest that these organs transmit photic information to the clock only in the morning and evening. Thus, different photoreceptors may be responsible for the different parts of a PRC (Kistenpfennig, 2012).

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

In nature, both daily light:dark cycles and temperature fluctuations are used by organisms to synchronize their endogenous time with the daily cycles of light and temperature. Proper synchronization is important for the overall fitness and wellbeing of animals and humans, and although a lot is known about light synchronization, this is not the case for 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. This study investigated whether different groups of the Drosophila clock neurons that regulate behavioral rhythmicity contribute to temperature synchronization at different absolute temperatures. Using spatially restricted expression of the clock gene period, this study shows that dorsally located clock neurons mainly mediate synchronization to higher (20°C:29°C) and ventral clock neurons to lower (16°C:25°C) temperature cycles. Molecularly, the blue-light photoreceptor Cryptochrome (Cry) dampens temperature-induced Period (Per)-Luciferase oscillations in dorsal clock neurons. Consistent with this finding, this study shows that in the absence of Cry very limited expression of Per in a few dorsal clock neurons is able to mediate behavioral temperature synchronization to high and low temperature cycles independent of light. This study shows that different subsets of clock neurons operate at high and low temperatures to mediate clock synchronization to temperature cycles, suggesting that temperature entrainment is not restricted to measuring the amplitude of such cycles. Cry dampens temperature input to the clock and thereby contributes to the integration of different Zeitgebers (Gentile, 2013).

This study has shown that different sets of clock neurons play a role for synchronization to low and high temperature cycles with identical amplitude. This shows that temperature entrainment does not solely rely on measurement of temperature differences but rather on measurement of absolute temperatures. This task is divided between different neuronal groups, opening the possibility that multiple temperature receptors -- expressed either in different clock neurons, other neurons in the brain, or in the PNS -- contribute to temperature entrainment. Cry seems to actively block the entrainment strength of temperature both at a molecular and behavioral level, which most likely contributes to Zeitgeber integration (Gentile, 2013).

Molecular evolution of a pervasive natural amino-acid substitution in Drosophila cryptochrome

Genetic variations in circadian clock genes may serve as molecular adaptations, allowing populations to adapt to local environments. This study carried out a survey of genetic variation in Drosophila cryptochrome (cry), the fly's dedicated circadian photoreceptor. An initial screen of 10 European cry alleles revealed substantial variation, including seven non-synonymous changes. The SNP frequency spectra and the excessive linkage disequilibrium in this locus suggested that this variation is maintained by natural selection. Focus was placed on a non-conservative SNP involving a leucine-histidine replacement (L232H); this polymorphism is common, with both alleles at intermediate frequencies across 27 populations surveyed in Europe, irrespective of latitude. Remarkably, it was possible to reproduce this natural observation in the laboratory using replicate population cages where the minor allele frequency was initially set to 10%. Within 20 generations, the two allelic variants converged to approximately equal frequencies. Further experiments using congenic strains, showed that this SNP has a phenotypic impact, with variants showing significantly different eclosion profiles. At the long term, these phase differences in eclosion may contribute to genetic differentiation among individuals, and shape the evolution of wild populations (Pegoraro, 2014).


EVOLUTIONARY HOMOLOGS
Photolyases

The photolyase-blue-light photoreceptor family is composed of cyclobutane pyrimidine dimer (CPD) photolyases, (6-4) photolyases, and blue-light photoreceptors. CPD photolyase and (6-4) photolyase are involved in photoreactivation for CPD and (6-4) photoproducts, respectively. CPD photolyase is classified into two subclasses, class I and II, based on amino acid sequence similarity. Blue-light photoreceptors are essential light detectors for the early development of plants. The amino acid sequence of the receptor is similar to sequences in the photolyases, although the receptor does not show photoreactivation activity. To investigate the functional divergence within the family, the amino acid sequences of the proteins were aligned. The alignment suggested that the recognition mechanisms of the cofactors and the substrate of class I CPD photolyases are different from those of class II CPD photolyases. Phylogenetic trees based on the alignment were reconstructed. Phylogenetic analysis suggests that the ancestral gene of the family had encoded CPD photolyase and that the gene duplication of the ancestral proteins had occurred at least eight times before the divergence between eubacteria and eukaryotes (Kanai, 1997).

Photolyase repairs ultraviolet (UV) damage to DNA by splitting the cyclobutane ring of the major UV photoproduct: the cis, syn-cyclobutane pyrimidine dimer (Pyr <> Pyr). The reaction is initiated by blue light and proceeds through long-range energy transfer, single electron transfer, and enzyme catalysis by a radical mechanism. The three-dimensional crystallographic structure of DNA photolyase from Escherichia coli is presented and the atomic model has been refined to an R value of 0.172 at 2.3 A resolution. The polypeptide chain of 471 amino acids is folded into an amino-terminal alpha/beta domain resembling dinucleotide binding domains and a carboxyl-terminal helical domain; a loop of 72 residues connects the domains. The light-harvesting cofactor 5,10-methenyltetrahydrofolylpolyglutamate (MTHF) binds in a cleft between the two domains. Energy transfer from MTHF to the catalytic cofactor flavin adenine dinucleotide (FAD) occurs over a distance of 16.8 A. The FAD adopts a U-shaped conformation between two helix clusters in the center of the helical domain and is accessible through a hole in the surface of this domain. Dimensions and polarity of the hole match those of a Pyr <> Pyr dinucleotide, suggesting that the Pyr <> Pyr "flips out" of the helix to fit into this hole, and that electron transfer between the flavin and the Pyr <> Pyr occurs over van der Waals contact distance (Park, 1995).

Employing UV-irradiated Drosophila melanogaster embryos as a source of material, three new proteins that selectively bind to UV-damaged DNA were identified and purified through to near homogeneity. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrates that it is a monomer protein that is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrates that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. Also discussed is a possible role for these DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products (Kai, 1995).

Animal-type photolyases have very limited sequence homology to microbial-type photolyases. Do animal and microbial enzymes have different or similar biochemical and photochemical properties? In particular, the chromophore/cofactor composition of animal photolyases is of special interest since neither the presence nor the nature of a second chromophore in these enzymes is known, in contrast to the microbial photolyases which contain FAD cofactor, and folate or deazaflavin as second chromophores. The Drosophila photolyase was produced in E. coli using the cloned gene. The enzyme contains FAD and folate and thus belongs in the folate class of enzymes but with an action spectrum peak at 420 nm (Kim, 1996).

Ultraviolet light (UV)-induced DNA damage can be repaired by DNA photolyase in a light-dependent manner. Two types of photolyase are known, one specific for cyclobutane pyrimidine dimers (CPD photolyase) and another specific for pyrimidine (6-4) pyrimidone photoproducts[(6-4)photolyase]. In contrast to the CPD photolyase, which has been detected in a wide variety of organisms, the (6-4)photolyase has been found only in Drosophila melanogaster. In the present study a gene encoding the Drosophila(6-4)photolyase was cloned, and the deduced amino acid sequence of the product was found to be similar to the CPD photolyase and to the blue-light photoreceptor of plants. A homolog of the Drosophila (6-4)photolyase gene was also cloned from human cells (Todo, 1996).

Highly purified D-DDB P1, an ultraviolet light (UV) damage-specific DNA-binding protein from Drosophila melanogaster, has been obtained from Drosophila Kc cells and found to be a nuclease. D-DDB P1 can selectively bind to pyrimidine (6-4) pyrimidone photoproducts, and in the presence of Mg++, D-DDB P1 can catalyze an incision immediately on the 3' and 5' sides of the (6-4) photoproduct site (Kai, 1998).

The (6-4)photoproduct DNA photolyase [(6-4)photolyase] repairs UV-induced pyrimidine (6-4) pyrimidone photoproduct [(6-4)photoproduct, pyr(6,4)pyr] in a light dependent manner. Drosophila (6-4)photolyase was purified to near homogeneity from Drosophila embryonic cells and is a 62 kDa protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified (6-4)photolyase repairs (6-4)photoproducts induced at 5'-CC-3' site (C[6,4]C) as well as T[6,4]T and T[6,4]C. Photoreactivation of (6-4)photoproduct constructed in M13 phage eliminates the replication block and abolishes induced mutagenesis in E. coli cells, suggesting that the (6-4)photolyase repairs the photoproduct to the unmodified form (Todo, 1998).

Yeast and many other organisms use nucleotide excision repair (NER) and photolyase in the presence of light (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs), a major class of DNA lesions generated by UV light. To study the role of photoreactivation at the chromatin level in vivo, yeast strains were used that contain minichromosomes (YRpTRURAP, YRpCS1) with well-characterized chromatin structures. The strains were either proficient (RAD1) or deficient (rad1 delta) in NER. In contrast to NER, photolyase rapidly repairs CPDs in non-nucleosomal regions, including promoters of active genes (URA3, HIS3, DED1) and in linker DNA between nucleosomes. CPDs in nucleosomes are much more resistant to photoreactivation. These results demonstrate a direct role of chromatin in modulation of a DNA repair process and an important role of photolyase in repair of damaged promoters with presumptive effects on gene regulation. In addition, photoreactivation provides an in vivo test for chromatin structure and stability. In active genes (URA3, HIS3), photolyase repairs the non-transcribed strand faster than the transcribed strand and can match fast removal of lesions from the transcribed strand by NER (transcription-coupled repair). Thus, the combination of both repair pathways ensures efficient repair of active genes (Suter, 1997).

The photolyase/blue-light photoreceptor family of proteins includes cyclobutane pyrimidine dimer photolyase, (6-4) photolyase and blue-light photoreceptors that were recently discovered in Arabidopsis thaliana, Sinapis alba and Chlamydomonas reinhardtii. Recently, two human genes, hCRY1 and hCRY2, have been identifed as members of this same family. The proteins encoded by these genes have no DNA repair activity and therefore are hypothesized to function in human blue-light response reactions. To identify downstream targets for these putative blue-light photoreceptors, interacting proteins were sought by the yeast two-hybrid method. The tetratricopeptide repeat protein 1, Tpr1, and the protein serine/threonine phosphatase 5 (PP5) that contains the TPR motif specifically interacts with hCRY2. The effect of the hCRY2-PP5 interaction on the protein phosphatase activity was investigated. hCRY2, but not the highly homologous (6-4) photolyase, is found to inhibit the phosphatase activity of PP5. This inhibition may be on the pathway of blue-light signal transduction reaction in humans (Zhao, 1997).

Blue-light photoreceptors of plants

Specific responses to blue light are found throughout the biological kingdom. These responses (which in higher plants include phototropism, inhibition of hypocotyl elongation, and stomatal opening) are in many cases thought to be mediated by flavin-type photoreceptors. But no such blue-light photoreceptor has yet been identified or isolated, although blue-light responses in plants were reported by Darwin over a century ago, long before the discovery of the now relatively well characterized red/far-red light photoreceptor, phytochrome. Here the isolation of a gene corresponding to the HY4 locus of Arabidopsis thaliana is described. The hy4 mutant is one of several mutants that are selectively insensitive to blue light during the blue-light-dependent inhibition of hypocotyl elongation response, which suggests that they lack an essential component of the cryptochrome-associated light-sensing pathway. The HY4 gene, isolated by gene tagging, encodes a protein with significant homology to microbial DNA photolyases.Because photolyases comprise a rare class of flavoproteins that catalyse blue-light-dependent reactions, the protein encoded by HY4 has a structure consistent with that of a flavin-type blue-light photoreceptor (Ahmad, 1993).

A more recent paper by Ahmad (1995) reports the characterization of novel mutations within the Arabidopsis thaliana HY4 gene, which has previously been shown to encode a protein (CRY1) with characteristics of a blue-light photoreceptor. Several point mutations were identified within the amino-terminal domain of CRY1: this region of CRY1 has high homology to photolyase and is likely to be involved in blue-light-mediated electron transfer. Mutations were found within the region of homology to the known chromophore binding domains of photolyase. Point mutations within the 200 amino acid carboxy-terminal extension distinguishing CRY1 from photolyase, likewise disrupt function of the protein. CRY1 was originally defined as the photoreceptor responsible for blue-light-mediated inhibition of hypocotyl elongation and it is now reported that anthocyanin accumulation in germinating seedlings is an additional phenotype under the control of this photoreceptor. This is shown to be mediated in part by modulation of mRNA levels of chalcone synthase, one of the anthocyanin biosynthetic enzymes. The effect of the novel mutations on both inhibition of hypocotyl elongation and anthocyanin biosynthesis have been evaluated: mutations with less severe effects on hypocotyl elongation show a similarly reduced effect on anthocyanin biosynthesis. These results are consistent with the cryptochrome photoreceptor mediating multiple regulatory pathways by the same primary mode of action (Ahmad, 1995).

Blue-light responses in higher plants are mediated by specific photoreceptors, which are thought to be flavoproteins; one such flavin-type blue-light receptor, CRY1 (for cryptochrome), which mediates inhibition of hypocotyl elongation and anthocyanin biosynthesis, has recently been characterized. Prompted by classical photobiological studies suggesting possible co-action of the red/far-red absorbing photoreceptor phytochrome with blue-light photoreceptors in certain plant species, the role of phytochrome in CRY1 action in Arabidopsis was investigated. The activity of the CRY1 photoreceptor can be substantially altered by manipulating the levels of active phytochrome (Pfr) with red or far-red light pulses subsequent to blue-light treatments. Furthermore, analysis of severely phytochrome-deficient mutants shows that CRY1-mediated blue-light responses are considerably reduced, even though Western blots confirm that levels of CRY1 photoreceptor are unaffected in these phytochrome-deficient mutant backgrounds. It is concluded that CRY1-mediated inhibition of hypocotyl elongation and anthocyanin production requires active phytochrome for full expression, and that this requirement can be supplied by low levels of either phyA or phyB (Ahmad, 1997).

A cDNA from Arabidopsis thaliana similar to microbial photolyase genes, and designated AT-PHH1, was isolated using a photolyase-like cDNA from Sinapsis alba (SA-PHR1) as a probe. Multiple isolations yielded only PHH1 cDNAs, and a few blue-light-receptor CRY1 (HY4) cDNAs (also similar to microbial photolyase genes), suggesting the absence of any other highly similar Arabidopsis genes. The AT-PHH1 and SA-PHR1 cDNA sequences predict 89% identity at the protein level, except for an AT-PHH1 C-terminal extension (111 amino acids), also not seen in microbial photolyases. AT-PHH1 and CRY1 show less similarity, including respective C-terminal extensions that are themselves mostly dissimilar. Analysis of fifteen AT-PHH1 genomic isolates reveals a single gene, with three introns in the coding sequence and one in the 5'-untranslated leader. Full-length AT-PHH1, and both AT-PHH1 and AT-PHH1 delta C-513 (truncated to be approximately the size of microbial photolyase genes) cDNAs, were overexpressed, respectively, in yeast and Escherichia coli mutants hypersensitive to ultraviolet light. The absence of significant effects on resistance suggests either that any putative AT-PHH1 DNA repair activity requires cofactors/chromophores not present in yeast or E. coli, or that AT-PHH1 encodes a blue-light/ultraviolet-A receptor rather than a DNA repair protein (Hoffman, 1996).

Phototropism (bending toward the light) is one of the best known plant tropic responses. Despite being reported by Darwin and others over a century ago to be specifically under the control of blue light, the photoreceptors mediating phototropism have remained unknown. A blue-light photoreceptor from Arabidopsis, named CRY1 for cryptochrome 1 has been characterized; this photoreceptor is a flavoprotein that mediates numerous blue-light-dependent responses. In Arabidopsis, HY4 (the gene encoding CRY1) is a member of a small gene family that also encodes a related photoreceptor, CRY2, which shares considerable functional overlap with CRY1. Mutant plants lacking both the CRY1 and the CRY2 blue-light photoreceptors are deficient in the phototropic response. Transgenic Arabidopsis plants overexpressing CRY1 or CRY2 show enhanced phototropic curvature. It is concluded that cryptochrome is one of the photoreceptors mediating phototropism in plants (Ahmad, 1998a).

A blue light (cryptochrome) photoreceptor from Arabidopsis, cry1, has been identified recently and shown to mediate a number of blue light-dependent phenotypes. Similar to phytochrome, the cryptochrome photoreceptors are encoded by a gene family of homologous members with considerable amino acid sequence similarity within the N-terminal chromophore binding domain. The two members of the Arabidopsis cryptochrome gene family (CRY1 and CRY2) overlap in function, but their proteins differ in stability: cry2 is rapidly degraded under light fluences (green, blue, and UV) that activate the photoreceptor, but cry1 is not. It is demonstrated by overexpression in transgenic plants of cry1 and cry2 fusion constructs that their domains are functionally interchangeable. Hybrid receptor proteins mediate functions similar to cry1 and include inhibition of hypocotyl elongation and blue light-dependent anthocyanin accumulation; differences in activity appear to be correlated with differing protein stability. Because cry2 accumulates to high levels under low-light intensities, it may have greater significance in wild-type plants under conditions when light is limited (Ahmad, 1998b).

Plants have at least two major photosensory receptors: phytochrome (absorbing primarily red/far-red light) and cryptochrome (absorbing blue/UV-A light); considerable physiological and genetic evidence suggests some form of communication or functional dependence between the receptors. Arabidopsis CRY1 and CRY2 (cryptochrome) are substrates for phosphorylation by a phytochrome A-associated kinase activity. Several mutations within the CRY1 C terminus lead to reduced phosphorylation by phytochrome preparations in vitro. Yeast two-hybrid interaction studies using expressed C-terminal fragments of CRY1 and phytochrome A from Arabidopsis confirm a direct physical interaction between both photoreceptors. In vivo labeling studies and specific mutant alleles of CRY1, which interfere with the function of phytochrome, suggest the possible relevance of these findings in vivo (Ahmad, 1998c).

Cryptochromes are a group of flavin-type blue light receptors that regulate plant growth and development. The function of Arabidopsis cryptochrome 2 in the early photomorphogenesis of seedlings was studied by using transgenic plants overexpressing CRY2 protein, and cry2 mutant plants accumulating no CRY2 protein. Cryptochrome 2 mediates blue light-dependent inhibition of hypocotyl elongation and stimulation of cotyledon opening under low intensities of blue light. In contrast to CRY1, the expression of CRY2 is rapidly down-regulated by blue light in a light-intensity dependent manner, which provides a molecular mechanism to explain at least in part that cryptochrome 2 functions primarily under low light during the early development of seedlings (Lin, 1998).

The shift in plants from vegetative growth to floral development is regulated by red-far-red light receptors (phytochromes) and blue-ultraviolet A light receptors (cryptochromes). A mutation in the Arabidopsis thaliana CRY2 gene encoding a blue-light receptor apoprotein (CRY2) is allelic to the late-flowering mutant, fha. Flowering in cry2/fha mutant plants is only incompletely responsive to photoperiod. Cryptochrome 2 (cry2) is a positive regulator of the flowering-time gene CO, the expression of which is regulated by photoperiod. Analysis of flowering in cry2 and phyB mutants in response to different wavelengths of light indicates that flowering is regulated by the antagonistic actions of phyB and cry2 (Guo, 1998).

Cryptochrome blue light photoreceptors share sequence similarity to photolyases, flavoproteins that mediate light-dependent DNA repair. However, cryptochromes lack photolyase activity and are characterized by distinguishing C-terminal domains. The signaling mechanism of Arabidopsis cryptochrome is mediated through the C terminus. On fusion with beta-glucuronidase (GUS), both the Arabidopsis CRY1 C-terminal domain (CCT1) and the CRY2 C-terminal domain (CCT2) mediate a constitutive light response. This constitutive photomorphogenic (COP) phenotype is not observed for mutants of cct1 corresponding to previously described cry1 alleles. It is proposed that the C-terminal domain of Arabidopsis cryptochrome is maintained in an inactive state in the dark. Irradiation with blue light relieves this repression, presumably through an intra- or intermolecular redox reaction mediated through the flavin bound to the N-terminal photolyase-like domain (Yang, 2000).

It has been assumed that cryptochromes function in a manner analogous to photolyases, mediating a light-driven redox reaction. Such a reaction could be either intermolecular or intramolecular. In the simplest form of the 'intermolecular model', cryptochrome activates a signaling partner, bound to the C teminus. In this model, which is very similar to the mode of action of photolyases, the C terminus undergoes no change as a result of irradiation. It is the bound signaling partner that undergoes a redox-induced change, with the function of CCT simply being to bind this partner. From the results that have been presented here, this model is wrong, as the 'isolated' CCT clearly displays properties distinct from those associated with CRY present in dark-grown seedlings. Furthermore, since these properties of the CCT are strikingly similar to those of light-activated CRY, and are not displayed by mutants of CCT, it is presumed that they are physiologically meaningful (Yang, 2000).

An alternative version of the 'intermolecular model', in keeping with the observations made in this study, postulates that upon light-induced activation, a regulatory molecule (Y) is released from its complex with CRY, thus exposing CCT and enabling binding to the signaling partner X. This model is similar in some ways to the regulation of protein kinase A, or glucocorticoid receptor, where ligand binding results in a change in conformation and release of regulatory subunits. For this model to hold, CRY would need to be fully associated in the dark with such a presumptive negative regulatory subunit (Yang, 2000).

In contrast to photolyases, cryptochrome may function through an intramolecular redox reaction, resulting in a change in the chemical properties of the CCT. If the catalytic form of the flavin in CRY is in the form of FADH-, as it is in photolyases, then the most likely acceptor for a light-induced redox change is a cysteine residue. CCT1 does not contain any cysteine residues, and therefore such an intramolecular redox reaction would need to be restricted to the N-terminal photolyase-like domain of CRY1. Alternatively, the catalytically active form of the flavin for CRY may be FAD or the semiquinone (FADH.), which for CRY1 has been demonstrated to be surprisingly stable. The significance of this point is that the flavin in either of these redox states may act as an electron acceptor, and a tryptophan residue (for example) could serve as the corresponding donor (Yang, 2000).

Somewhat irrespective of the details of the light-induced activation of CCT, the data indicate that CRY function is achieved through a signaling event mediated through the C terminus. This conclusion follows from the findings concerning the activity of the GUS-CCT transgenes and is in keeping with observations regarding mutant cry1 alleles containing lesions within the C terminus. This signaling likely involves interaction of CCT with another protein(s) and the identity of such a protein is obviously of interest (Yang, 2000).

Many of the properties of Arabidopsis seedlings expressing the CCT transgenes are reminiscent of mutants that affect either COP1 or the COP9 complex. Mutations in COP1, a zinc finger protein, result in short hypocotyls in the dark, and abnormal gene expression and chloroplast development, similar to those described here for the CCT-expressing plants. Whereas many alleles of cop1 are sterile, weak alleles are fertile and flower early, again similar in these respects to the CCT plants. Many of these properties of cop1 alleles are shared by mutants affecting other components of the COP9 signalosome, a multisubunit complex that has homologies to animal proteins and is believed to play a role in protein degradation. The COP9 complex is required for COP1 accumulation, which in turn negatively regulates photomorphogenesis. This latter step involves HY5, a bZIP DNA binding protein that physically interacts with COP1. HY5 levels are increased in the light, apparently a result of COP1 translocation from the nucleus to the cytoplasm (Yang, 2000 and references therein).

In view of the similarity of the mutant plants expressing CCT and both the COP1 mutants and mutants of the subunits of the COP9 complex, the following is considered as a likely explanation for the CCT phenotype: subsequent to the redox-mediated activation of the C terminus, it is proposed that CCT binds to one of four possible types of signaling partners. In the first option, CRY interacts in a negative manner with COP1, in this manner phenocopying loss-of-function cop1 alleles. In a similar manner, light-activated CRY may negatively interact with any one of the many COP9 signalosome subunits; again these interactions would phenocopy loss-of-function mutations affecting any of the subunits of this complex. Third, CRY may interact with signaling molecules downstream, or (fourth) upstream from the COP1 and the COP9 complex. In future studies, the details of the redox reaction and the identity of these CRY signaling partner(s) need to be identified. Furthermore, it will be of interest to see if the general properties described here for Arabidopsis CRY, are found to hold for animal cryptochromes, which, at least in the case of Drosophila CRY, also function as photoreceptors (Yang, 2000 and references therein).

Most organisms, from cyanobacteria to mammals, use circadian clocks to coordinate their activities with the natural 24-h light/dark cycle. The clock proteins of Drosophila and mammals exhibit striking homology but do not show similarity with clock proteins found so far from either cyanobacteria or Neurospora. Each of these organisms uses a transcriptionally regulated negative feedback loop in which the messenger RNA levels of the clock components cycle over a 24-h period. Proteins containing PAS domains are invariably found in at least one component of the characterized eukaryotic clocks. This study describes ADAGIO1 (ADO1), a gene of Arabidopsis thaliana that encodes a protein containing a PAS domain. A loss-of-function ado1 mutant is altered in both gene expression and cotyledon movement in circadian rhythmicity. Under constant white or blue light, the ado1 mutant exhibits a longer period than that of wild-type Arabidopsis seedlings, whereas under red light cotyledon movement and stem elongation are arrhythmic. Both yeast two-hybrid and in vitro binding studies show that there is a physical interaction between ADO1 and the photoreceptors CRY1 and phyB (phytochromes are red/far-red light-absorbing receptors). It is proposed that ADO1 is an important component of the Arabidopsis circadian system (Jarillo, 2001).

Cryptochromes (CRY) are blue light photoreceptors that mediate various light-induced responses in plants and animals. Arabidopsis CRY (CRY1 and CRY2) functions through negatively regulating constitutive photomorphogenic (COP) 1, a repressor of photomorphogenesis. Water evaporation and photosynthesis are regulated by the stomatal pores in plants, which are closed in darkness but open in response to blue light. There is evidence only for the phototropin blue light receptors (PHOT1 and PHOT2) in mediating blue light regulation of stomatal opening. This study reports previously uncharacterized role for Arabidopsis CRY and COP1 in the regulation of stomatal opening. Stomata of the cry1 cry2 double mutant showed reduced blue light response, whereas those of the CRY1-overexpressing plants showed hypersensitive response to blue light. In addition, stomata of the phot1 phot2 double mutant responded to blue light, but those of the cry1 cry2 phot1 phot2 quadruple mutant hardly responded. Strikingly, stomata of the cop1 mutant are constitutively open in darkness and stomata of the cry1 cry2 cop1 and phot1 phot2 cop1 triple mutants are open as wide as those of the cop1 single mutant under blue light. These results indicate that CRY functions additively with PHOT in mediating blue light-induced stomatal opening and that COP1 is a repressor of stomatal opening and likely acts downstream of CRY and PHOT signaling pathways (Mao, 2005).

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. This study demonstrates that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. Light-dependent changes in the cryptochrome1 (cry1) protein was further characterized in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms (Bouly, 2007).

Cryptochrome in zebrafish

Zebrafish tissues and cells have the unusual feature of not only containing a circadian clock, but also being directly light-responsive. Several zebrafish genes are induced by light, but little is known about their role in clock resetting or the mechanism by which this might occur. This study shows that Cryptochrome 1a (Cry1a) plays a key role in light entrainment of the zebrafish clock. Intensity and phase response curves reveal a strong correlation between light induction of Cry1a and clock resetting. Overexpression studies show that Cry1a acts as a potent repressor of clock function and mimics the effect of constant light to 'stop' the circadian oscillator. Yeast two-hybrid analysis demonstrates that the Cry1a protein interacts directly with specific regions of core clock components, CLOCK and BMAL, blocking their ability to fully dimerize and transactivate downstream targets, providing a likely mechanism for clock resetting. A comparison of entrainment of zebrafish cells to complete versus skeleton photoperiods reveals that clock phase is identical under these two conditions. However, the amplitude of the core clock oscillation is much higher on a complete photoperiod, as are the levels of light-induced Cry1a. It is believed that Cry1a acts on the core clock machinery in both a continuous and discrete fashion, leading not only to entrainment, but also to the establishment of a high-amplitude rhythm and even stopping of the clock under long photoperiods (Tamai, 2007).

Blue-light photoreceptors of mammals
Blue-light photoreceptors of mammals

Recently, a human cDNA clone with high sequence homology to the photolyase/blue-light photoreceptor family was identified. The putative protein encoded by this gene exhibits a strikingly high (48% identity) degree of homology to the Drosophila melanogaster (6-4) photolyase. A second human gene has been identified whose amino acid sequence displays 73% identity to the first one. The two genes have been named CRY1 and CRY2, respectively. The corresponding proteins hCRY1 and hCRY2 were purified and characterized as maltose-binding fusion proteins. Similar to other members of the photolyase/blue-light photoreceptor family, both proteins were found to contain FAD and a pterin cofactor. Like the plant blue-light photoreceptors, both hCRY1 and hCRY2 lack photolyase activity on the cyclobutane pyrimidine dimer and the (6-4) photoproduct. It is concluded that these newly discovered members of the photolyase/photoreceptor family are not photolyases and instead may function as blue-light photoreceptors in humans (Hsu, 1996).

Mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors, have been isolated and characterized. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. Antibodies specific against each gene product were raised using their C-terminal sequences, which differ completely between the genes. Immunofluorescent staining of cultured mouse cells reveals that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. Using green fluorescent protein fused peptides it has been demonstrated that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 contains the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells show a tight binding activity to DNA Sepharose, while CRY2 protein does not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that, despite the similarity in the sequence, the two proteins have distinct functions (Kobayashi, 1998).

In mammals the retina contains photoactive molecules responsible for both vision and circadian photoresponse systems. Opsins, which are located in rods and cones, are the pigments for vision but it is not known whether they play a role in circadian regulation. A subset of retinal ganglion cells with direct projections to the suprachiasmatic nucleus (SCN) are at the origin of the retinohypothalamic tract that transmits the light signal to the master circadian clock in the SCN. However, the ganglion cells are not known to contain rhodopsin or other opsins that may function as photoreceptors. Two blue-light photoreceptors, cryptochromes 1 and 2 (CRY1 and CRY2), recently discovered in mammals, are specifically expressed in the ganglion cell and inner nuclear layers of the mouse retina. In addition, CRY1 is expressed at high levels in the SCN and oscillates in this tissue in a circadian manner. These data, in conjunction with the established role of CRY2 in photoperiodism in plants, lead to a proposal that mammals have a vitamin A-based photopigment (opsin) for vision and a vitamin B2-based pigment (cryptochrome) for entrainment of the circadian clock (Miyamoto, 1998).

Molecular clocks do not oscillate with an exact 24-hour rhythmicity but are entrained to solar day/night rhythms by light. The mammalian proteins Cryl and Cry2, which are members of the family of plant blue-light receptors (cryptochromes) and photolyases, have been proposed as candidate light receptors for photoentrainment of the biological clock. Mice lacking the Cryl or Cry2 protein display accelerated and delayed free-running periodicity of locomotor activity, respectively. Strikingly, in the absence of both proteins, an instantaneous and complete loss of free-running rhythmicity is observed. This suggests that, in addition to a possible photoreceptor and antagonistic clock-adjusting function, both proteins are essential for the maintenance of circadian rhythmicity (van der Horst, 1999).

Cryptochrome (Cry), a photoreceptor for the circadian clock in Drosophila, binds to the clock component TIM in a light-dependent fashion and blocks its function. In mammals, genetic evidence suggests a role for Crys within the biological clock, distinct from hypothetical photoreceptor functions. Mammalian CRY1 and CRY2 have been shown to act as light-independent inhibitors of CLOCK-BMAL1, the activator driving Per1 transcription. CRY1 or CRY2 (or both) show light-independent interactions with CLOCK and BMAL1, as well as with PER1, PER2, and TIM. No systematic or substantive differences have been observed in the ability of hCRY1 or hCRY2 to inhibit CLOCK-BMAL1 in light and dark conditions. Thus, mammalian CRY proteins act as light-independent components of the circadian clock and probably regulate Per1 transcriptional cycling by contacting both the activator and its feedback inhibitors. The light-independent role of mammalian Cry proteins in circadian clock negative feedback contrasts sharply with that of Drosophila Cry, which has been demonstrated to function directly as a photoreceptor that regulates the action of the Per-Tim complex. It is suggested that Drosophila Cry exemplifies the ancestral role of a photoreceptor acting as a light-dependent regulator of the circadian feedback loop, whereas mammalian Crys have preserved a role within the circadian feedback loop but shed their direct photoreceptor function. The possibility cannot be excluded that mammalian Cry proteins act as photoreceptors for other possible functions, circadian or otherwise, not detected in these assays (Griffin, 1999).

Mice lacking mCry1 and mCry2 are behaviorally arrhythmic. mCry2 is rhythmically expressed in the SCN in a manner similar to mCry1. Cyclic expression of the clock genes mPer1 and mPer2 (mammalian Period genes 1 and 2) in the suprachiasmatic nucleus and peripheral tissues is abolished and mPer1 and mPer2 mRNA levels are constitutively high. These findings indicate that the biological clock is eliminated in the absence of both mCRY1 and mCRY2 and support the idea that mammalian CRY proteins act in the negative limb of the circadian feedback loop. The high mRNA levels of mPer1 and mPer2 in mCry1/mCry2-deficient mice suggest that mCRY proteins negatively affect mPer expression. The mCry double-mutant mice retain the ability to have mPer1 and mPer2 expression induced by a brief light stimulus known to phase-shift the biological clock in wild-type animals. Thus, mCRY1 and mCRY2 are dispensable for light-induced phase shifting of the biological clock. The data suggest that photoreceptors other than mCRY proteins and rod/cone opsins [for example, mammalian homologs of the recently discovered fish VA-opsin or Xenopus laevis melanopsin] may be responsible for photic entrainment. Although mPer transcription repression by mCRY proteins (and thus their function in core oscillation) is light-independent, the possibliity that mCRY proteins may act as photoreceptor proteins cannot be completely ruled out: (1) it is possible that phase-shifting is mediated via more than one photoreceptor system, implying functional redundancy; (2) mCRY proteins may be involved in transmitting light inputs to the clock other than those required for phase shifting, such as changes in the length of the day and night, and information on dusk and dawn. Future experiments should shed light on the mysterious blue-light receptor properties of mCRY proteins (Okamura, 1999).

Cryptochromes regulate the circadian clock in animals and plants. Humans and mice have two cryptochrome (Cry) genes. Mice lacking the Cry2 gene have reduced sensitivity to acute light induction of the circadian gene mPer1 in the suprachiasmatic nucleus (SCN) and have an intrinsic period 1 hr longer than normal. In this study, Cry1-/- and Cry1-/-Cry2-/- mice were generated and their circadian clocks were analyzed at behavioral and molecular levels. Behaviorally, the Cry1-/- mice have a circadian period 1 hr shorter than wild type and the Cry1-/-Cry2-/- mice are arrhythmic in constant darkness (DD). Biochemically, acute light induction of mPer1 mRNA in the SCN is blunted in Cry1-/- and abolished in Cry1-/-Cry2-/- mice. In contrast, the acute light induction of mPer2 in the SCN was intact in Cry1-/- and Cry1-/-Cry2-/- animals. Importantly, in double mutants, mPer1 expression is constitutively elevated and no rhythmicity is detected in either 12-hr light/12-hr dark or DD, whereas mPer2 expression appears rhythmic in 12-hr light/12-hr dark, but nonrhythmic in DD with intermediate levels. These results demonstrate that Cry1 and Cry2 are required for the normal expression of circadian behavioral rhythms, as well as circadian rhythms of mPer1 and mPer2 in the SCN. The differential regulation of mPer1 and mPer2 by light in Cry double mutants reveals a surprising complexity in the role of cryptochromes in mammals (Vitaterna, 1999).

The role of mPer1 and mPer2 in regulating circadian rhythms was assessed by disrupting these genes. Mice homozygous for the targeted allele of either mPer1 or mPer2 have severely disrupted locomotor activity rhythms during extended exposure to constant darkness. Clock gene RNA rhythms are blunted in the suprachiasmatic nucleus of mPer2 mutant mice, but not in mPER1-deficient mice. Peak mPER and mCRY1 protein levels are reduced in both lines. Behavioral rhythms of mPer1/mPer3 and mPer2/mPer3 double-mutant mice resemble rhythms of mice with disruption of mPer1 or mPer2 alone, respectively, confirming the placement of mPer3 outside the core circadian clockwork. In contrast, mPer1/mPer2 double-mutant mice are immediately arrhythmic. Thus, mPER1 influences rhythmicity primarily through interaction with other clock proteins, while mPER2 positively regulates rhythmic gene expression, and there is partial compensation between products of these two genes (Bae, 2001).

To assess the impact of targeted disruption of mPer1 on molecular rhythms, patterns of gene expression in the SCN were examined on the first day in DD. Rhythmic expression of mPer2, mCry1, and Bmal1 RNAs is unaltered in the SCN of mPER1-deficient mice. In contrast to the lack of effect on SCN gene expression, clock protein rhythms in the SCN are markedly altered in mPER1-deficient mice. In wild-type mice, robust rhythms of nuclear mPER1, mPER2, and mCRY1 were detected on the first day in DD. mPER1 staining is absent in mPER1-deficient mice (Bae, 2001).

Gene expression rhythms are markedly altered in mPer2 mutant mice. In the SCN of mice homozygous for the mPer2 mutation, mPer1, mCry1 levels are rhythmic, while mPer2 levels are not. Bmal1 RNA levels vary significantly with time, but the data are not sinusoidal in shape, e.g., not rhythmic per se. There is a significant main effect of circadian time for each of the four genes examined, as well as a significant effect of genotype (mCry1, Bmal1) or a significant interaction (mPer1, mPer2). Post-hoc, pairwise comparisons revealed that mice homozygous for the mPer2 mutation have significantly depressed peak levels of mPer1, mPer2, mCry1, and Bmal1 gene expression in the SCN. These results are consistent with previous studies showing reduced levels of gene expression in the SCN of mPer2 mutant mice and further support a role for mPER2 as a positive regulator within the circadian feedback loop (Bae, 2001).

Posttranslational regulation of clock proteins in mouse liver has been examined in vivo. The mouse PERIOD proteins (mPER1 and mPER2), CLOCK, and BMAL1 undergo robust circadian changes in phosphorylation. These proteins, the cryptochromes (mCRY1 and mCRY2), and casein kinase I epsilon (CKIepsilon) form multimeric complexes that are bound to DNA during negative transcriptional feedback. CLOCK:BMAL1 heterodimers remain bound to DNA over the circadian cycle. The temporal increase in mPER abundance controls the negative feedback interactions. Analysis of clock proteins in mCRY-deficient mice shows that the mCRYs are necessary for stabilizing phosphorylated mPER2 and for the nuclear accumulation of mPER1, mPER2, and CKIepsilon. in vivo evidence is provided that casein kinase I delta is a second clock relevant kinase (Lee, 2001).

mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop

Two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In other words, mCRY1/mPER and mCRY2/mPER dimers inhibit CLOCK:BMAL1-mediated transcriptional activation, thus resulting in the negative regulation of PER and TIM in the nucleus. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1-mediated transcription. When mPER3 is coexpressed with either mPER1 or mPER2, mPER3 is dramatically redistributed from cytoplasm to become expressed in both cytoplasm and nucleus. mPER1 is more effective than mPER2 in promoting nuclear entry of mPER3; that is, nucleus-only location was found in 3× more cells with mPER1 cotransfections, as compared with mPER2. Despite trying all possible combinations of mPER proteins with mTIM, including adding all four proteins at once, a nucleus-only location of mPER1 or mPER2 could not be induced in greater than 30% of NIH3T3 cells. This differs dramatically from the in vivo situation in which both mPER1 and mPER2 are entirely nuclear in SCN cells when detectable. Thus, it would appear that mPER function cannot be fully reconstituted in NIH3T3 cells. Since mTim fails to drive mPER nuclear localization, it is concluded that the mTim function is not conserved between flies and mammals. This suggests that there are other clock-relevant factors important for the nuclear translocation of the mPER proteins (Kume, 1999).

This suggests cryptochrome might be involved in the negative limb of the feedback loop that results in the lowering of transcriptional levels of clock related genes. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially (Kume, 1999).

In addition to a potential direct inhibitory effect of the mCRY proteins on the CLOCK:BMAL1-E box complex, the cryptochromes could also inhibit transcription by directly interacting with the mPER proteins and translocating them to the nucleus for subsequent transcriptional effects. To evaluate the potential for protein-protein interactions between the mCRY and mPER families, coimmunoprecipitation using epitope-tagged proteins was carried out. COS7 cells cotransfected with expression plasmids encoding mCRY1-HA and either mPER1-V5, mPER2-V5, mPER3-V5, or mTIM-V5 expressed each V5-tagged protein prior to immunoprecipitation. Immunoprecipitation with the HA antibody and analysis of the immunoprecipitated material with anti-V5 antibodies indicates the presence of heterodimeric interactions between mCRY1 and each of the mPER and mTIM proteins. Coimmunoprecipitation experiments using mCRY2-HA instead of mCRY1-HA similarily show the presence of heterodimeric interactions between mCRY2 and each of the mPER and mTIM proteins. Having shown that mCRY:mPER heterodimers could exist, it was next determined whether such interactions translocate the mPER proteins to the nucleus. In marked contrast to the lack of effect of any pairwise combination of mPER:mPER or mPER:mTIM interactions to translocate mPER1 and mPER2 to the nucleus, each mCRY protein profoundly changes the location of all three mPER proteins in NIH3T3 and COS7 cells. This is most apparent for mPER1 and mPER2, which are almost entirely nuclear after cotransfection with either mCRY1 or mCRY2. Curiously, each mCRY protein changes mPER3 from mainly cytoplasm only (>80%) to both cytoplasm and nucleus (>70%) to a degree similar to that induced by cotransfection of mPER3 with mPER1. However, when mPER3 is cotransfected with mPER1 and either mCRY1 or mCRY2, each of the three protein combinations change mPER3's location from 13%-20% exclusively nuclear location to an exclusively nuclear location in a majority of cells (Kume, 1999).

These data indicate that the mCRY proteins can heterodimerize with the mPER proteins and mTIM and that mCRY:mPER interactions mimic the in vivo situation, that is, the almost complete translocation of mPER1 and mPER2 to the nucleus. Moreover, trimeric interactions among the mPER and mCRY proteins appear necessary for complete nuclear translocation of mPER3. The data also suggest that the nuclear translocation of the mPER proteins is dependent on mCRY1 and mCRY2. The mCRY proteins, however, appear to be able to translocate to the nucleus independent of the mPERs. Even with massive overexpression of mCRY proteins in cell culture, they are always greater than 90% nuclear. If a PER partner were required for CRY nuclear translocation, a high CRY:PER ratio should result in cytoplasmic trapping of CRY. This was not observed (Kume, 1999).

The discovery of the functions of mCRY1 and mCRY2 within the clock feedback loop provides a sharper view of the molecular working of the mammalian clock. The cloning of a family of three mPer genes over the past 2 years has added to understanding of the negative limb of a mammalian clock feedback loop. But close examination of these putative clock elements and mTim has shown that they alone cannot fully explain the negative limb of the feedback loop. It thus seemed likely that other factors were involved. mCRY1 and mCRY2 have been shown to be major players in the negative limb of the clock feedback loop. These data also explain the strong loss-of-function phenotype of mCry1-/-mCry2-/- mice (Kume, 1999).

The cell culture data show that the mCRY proteins function as dimeric and potentially trimeric partners for the mPER proteins and that these interactions lead to the nuclear translocation and/or retention of the mPER proteins. This is in marked contrast to the inability of mTIM to translocate the three mPER proteins to the nucleus in cell culture and the invariant nature of endogenous mTIM levels in the nuclei of SCN neurons; mTIM immunoreactivity is present in the nucleus of most SCN neurons at all times throughout the circadian cycle. Thus, the mCRY proteins appear to function as nuclear translocators of the mPERs. In addition, mCRY nuclear translocation does not appear to be dependent on mPER:mCRY interactions. This is different from the situation in the fly in which Per:Tim heterodimers appear essential for the translocation of both Per and Tim to the nucleus (Kume, 1999).

The role of mTIM in the mammalian clock remains enigmatic. Even though mTIM does not appear to be important for the nuclear translocation of the mPER proteins, mTIM is localized to the nucleus in vivo, and it does cause a modest inhibition of CLOCK:BMAL1- and MOP4:BMAL1-mediated transcription in cell culture. In addition, mCRY1 and mCRY2 each appear capable of forming heterodimeric complexes with mTIM. Once in the nucleus, mTIM could therefore still have a role in modulating negative feedback of the mPER and/or mCRY1 rhythms. Another observation from these studies is the finding that the mCry1 gene forms its own interacting loop within the collective mammalian clock feedback mechanism. Evidence for this contention is substantial. mCry1 RNA and protein levels exhibit a circadian rhythm in the SCN; the RNA rhythm is dependent on a functional CLOCK protein, and the mCry1 promoter region contains a functional CACGTG E box. In fact, it is entirely possible that the mCry1 rhythm is the dominant oscillation in the mammalian clock feedback loop. This might explain the dominant circadian function of the mCry1 gene over mCry2, whose RNA levels do not oscillate. One normal mCry1 allele sustains normal circadian rhythms in behavior, while one mCry2 allele leads to arrhythmicity with increasing time in constant darkness. It is not known precisely how the mPER and mCRY proteins inhibit CLOCK:BMAL1-mediated transcription, but the data suggest differential sites of action. In the fly, multimeric complexes involving Per, Tim, and Clock appear to be important. It is thus possible that the mPER proteins, mTIM, and the mCRY proteins are all complexed with CLOCK. In addition, mCRY1 and mCRY2 appear to be capable of inhibiting E box-mediated transcription independent of CLOCK. This suggests that the mammalian cryptochromes also interact directly with either BMAL1 or the E box itself. Indeed, mCRY1 can bind tightly to dsDNA Sepharose. Even though the major components of the loop have been identified, the way in which a 24 hr time constant is incorporated into the mammalian clock loop has not been elucidated. Based on studies in Drosophila, posttranslational processes such as phosphorylation, proteosomal proteolysis, and gated nuclear entry are likely to contribute to the time delay. It is not yet known which component(s) of the loop is affected by these processes (Kume, 1999 and references).

In summary, the data show that mCRY1 and mCRY2 are redundant but still essential components of the negative limb of the clock feedback loop. The redundant function of these proteins explains the maintenance of circadian rhythmicity when either gene is deleted and explains the strong arrhythmic phenotype of double knockout mice. The different direction of period change in mCry1-/- versus mCry2-/- mice may result from differing affinities of these proteins for the mPER proteins or other clock components, and/or different levels of protein expression. It is predicted that the SCN of mCry1-/-mCry2-/- animals will show disrupted mPer RNA and protein rhythms with the mPER proteins stuck in the cytoplasm and mPer RNA levels at constant high values because of the absence of negative feedback. Placing the mammalian cryptochromes in the negative limb of the clock feedback loop sets forth a number of new hypotheses that can now be tested (Kume, 1999 and references).

Cycling of Cryptochrome proteins is not necessary for circadian-clock function in mammalian fibroblasts

An interlocked transcriptional-translational feedback loop (TTFL) is thought to generate the mammalian circadian clockwork in both the central pacemaker residing in the hypothalamic suprachiasmatic nuclei and in peripheral tissues. The core circadian genes, including Period1 and Period2 (Per1 and Per2), Cryptochrome1 and Cryptochrome2 (Cry1 and Cry2), Bmal1, and Clock are indispensable components of this biological clockwork. The cycling of the Per and Cry clock proteins has been thought to be necessary to keep the mammalian clock ticking. This study provides a novel cell-permeant protein approach for manipulating cryptochrome protein levels to evaluate the current transcription and translation feedback model of the circadian clockwork. Cell-permeant cryptochrome proteins appear to be functional on the basis of several criteria, including the abilities to (1) rescue circadian properties in Cry1−/−Cry2−/− mouse fibroblasts, (2) act as transcriptional repressors, and (3) phase shift the circadian oscillator in Rat-1 fibroblasts. By using cell-permeant cryptochrome proteins, it has been demonstrated that cycling of CRY1, CRY2, and BMAL1 is not necessary for circadian-clock function in fibroblasts. These results are not supportive of the current version of the transcription and translation feedback-loop model of the mammalian clock mechanism, in which cycling of the essential clock proteins CRY1 and CRY2 is thought to be necessary (Fan, 2007).

Is it possible that there is a core mammalian clockwork that is purely posttranslational? This possibility cannot be ignored, and it is possible that the results with CP-CRY proteins intimate that underlying posttranslational clock. An intriguing final speculation upon the discrepancy between these results and the earlier literature is that artificial intracellular expression of CRY1 has the effect of continuously flooding the cells with newly synthesized protein that is not posttranslationally modified, whereas the CP-CRYs have already incorporated the FAD/pterin cofactors and may be otherwise posttranslationally modified. Perhaps if a posttranslational oscillator is the core mammalian clockwork, the introduction of posttranslationally modified CRY (as with CP-CRYs) may perturb the core oscillator less than the new synthesis of unmodified CRY proteins. It may be in the fullness of time that this era of re-evaluation will lead to minor alterations of the understanding of the mammalian clockwork and that the transcriptional-translational feedback loop model will emerge triumphant. In contrast, this time of re-evaluation may lead to a very different comprehension of the mammalian clock mechanism (Fan, 2007).

Cryptochrome domain structure

Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. Nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells, have been examined. mCRY1 and mCRY2 have been shown to complex with PER proteins leading to nuclear import. Nuclear translocation of mPER1 and mPER2 (1) involves physical interactions with mPER3; (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2 double-deficient cells lacking a functional biological clock. Moreover, nuclear localization of endogenous mPER1 is observed in cultured mCry1/mCry2 double-deficient cells as well as in the liver and the suprachiasmatic nuclei (SCN) of mCry1/mCry2 double-mutant mice. This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions (i.e., in the intact mouse) in the absence of any CRY protein. The mPER3 amino acid sequence predicts the presence of a cytoplasmic localization domain (CLD) and a nuclear localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophila is conserved in mammals, but with the novel twist that mPER3 can act as the dimerizing partner (Yagita, 2000).

Mouse mCRY1 and zebrafish zCRY1a and zCRY3 belong to the DNA photolyase/Cryptochrome family. mCRY1 and zCRY1a repress CLOCK:BMAL1-mediated transcription, whereas zCRY3 does not. Reciprocal chimeras between zCRY1a and zCRY3 were generated to determine the zCRY1a regions responsible for nuclear translocation, interaction with the CLOCK:BMAL1 heterodimer, and repression of CLOCK:BMAL1-mediated transcription. Three regions, RD-2a-(126-196), RD-1-(197-263), and RD-2b-(264-293), were identified. Proteins in this family consist of an N-terminal alpha/beta domain and a C-terminal helical domain connected by an interdomain loop. RD-2a is within this loop, RD-1 is at the N-terminal 50 amino acids, and RD-2b at the following 31 amino acid residues of the helical domain. Either RD-2a or RD-1 is required for interaction with the CLOCK: BMAL1 heterodimer, and either RD-1 or RD-2b is required for the nuclear translocation of CRY. Both of these functions are prerequisites for the transcriptional repressor activity. The functional nuclear localizing signal in the RD-2b region also was identified. The sequence is well conserved among repressor-type CRYs, including mCRY1. Mutations in the nuclear localizing signal of mCRY1 reduce the extent of its nuclear localization. These findings show that both nuclear localization and interaction with the CLOCK:BMAL heterodimer are essential for transcriptional repression by CRY (Hirayama, 2003).

Circadian rhythms are driven by molecular clocks composed of interlocking transcription/translation feedback loops. Cryptochrome proteins are critical components of these clocks and repress the activity of the transcription factor heterodimer CLOCK/BMAL1. Unlike the homologous DNA repair enzyme 6-4 PHOTOLYASE, Cryptochromes have extended carboxyl-terminal tails and cannot repair DNA damage. Unlike mammals, Xenopus laevis contains both Cryptochromes (xCRYs) and 6-4 PHOTOLYASE (xPHOTOLYASE), providing an excellent comparative tool to study Cry repressive function. Findings can be extended to CRYs in general because xCRYs share high sequence homology with mammalian CRYs. Deletion of xCRYs' C-terminal domain produces proteins that are, like xPHOTOLYASE, unable to suppress CLOCK/BMAL1 activation. However, these truncations also cause the proteins to be cytoplasmically localized. A heterologous nuclear localization signal (NLS) restores the truncation mutants' nuclear localization and repressive activity. These results demonstrate that the CRYs' C termini are essential for nuclear localization but not necessary for the suppression of CLOCK/BMAL1 activation; this finding indicates that these two functions reside in separable domains. Furthermore, the functional differences between CRYs and PHOTOLYASE can be attributed to the few amino acid changes in the conserved portions of these proteins (Zhu, 2003).

Nucleocytoplasmic shuttling and mCRY-dependent inhibition of ubiquitylation of the mPER2 clock protein

The core oscillator generating circadian rhythms in eukaryotes is composed of transcription-translation-based autoregulatory feedback loops in which clock gene products negatively affect their own expression. A key step in this mechanism involves the periodic nuclear accumulation of clock proteins following their mRNA rhythms after ~6 h delay. Nuclear accumulation of mPER2 is promoted by mCRY proteins. Using COS7 cells and mCry1/mCry2 double mutant mouse embryonic fibroblasts transiently expressing GFP-tagged (mutant) mPER2, it has been shown that the protein shuttles between nucleus and cytoplasm using functional nuclear localization and nuclear export sequences. Moreover, evidence is provided that mCRY proteins prevent ubiquitylation of mPER2 and subsequent degradation of the latter protein by the proteasome system. Interestingly, mPER2 in turn prevents ubiquitylation and degradation of mCRY proteins. On the basis of these data a model is proposed in which shuttling mPER2 is ubiquitylated and degraded by the proteasome unless it is retained in the nucleus by mCRY proteins (Yagita, 2002).

One of the key features of the self-sustaining circadian feedback loop is the phase delay of several hours between mRNA and protein peaks for genes involved in the circadian core oscillator. A mechanism of nuclear-cytoplasmic shuttling and ubiquitin-proteasome-dependent degradation obviously adds another level at which this delay can be modulated. Examination of the predicted amino acid sequence of other clock proteins reveals the presence of three putative NES domains in mPER1 (residues 138-149, 489-498 and 981-988) and mPER3 (residues 54-63, 399-408 and 913-920), all with conservation of the critical hydrophobic amino acid residues. In addition, human BMAL1 also contains two potential NES sequences (residues 105- 114 and 124- 134). It remains to be determined whether the mechanism of nuclear- cytoplasmic shuttling in conjunction with the ubiquitin-proteasome system is restricted to the mPER2 protein or whether it extends to other clock proteins. Similarly, it is not known whether shuttling of mPER2 (and perhaps other clock proteins) in combination with ubiquitin-proteasome-mediated degradation is clock regulated or whether it is a general mechanism involved in protein stabilization. Given the parallel with other systems the latter option is favored. Ubiquitylation and proteasomal degradation of clock proteins by itself is not a new finding. Light-dependent proteasomal degradation of Timeless has been described in Drosophila. However, this is the first example of the (light-independent) involvement of the ubiquitin-proteasome pathway in the core mechanism of the circadian oscillator (Yagita, 2002).

Functional evolution of the photolyase/cryptochrome protein family: importance of the C terminus of mammalian CRY1 for circadian core oscillator performance

Cryptochromes are composed of a core domain with structural similarity to photolyase and a distinguishing C-terminal extension. While plant and fly CRYs act as circadian photoreceptors, using the C terminus for light signaling, mammalian CRY1 and CRY2 are integral components of the circadian oscillator. However, the function of their C terminus remains to be resolved. The C-terminal extension of mCRY1 harbors a nuclear localization signal and a putative coiled-coil domain that drives nuclear localization via two independent mechanisms and shifts the equilibrium of shuttling mammalian CRY1 (mCRY1)/mammalian PER2 (mPER2) complexes towards the nucleus. Importantly, deletion of the complete C terminus prevents mCRY1 from repressing CLOCK/BMAL1-mediated transcription, whereas a plant photolyase gains this key clock function upon fusion to the last 100 amino acids of the mCRY1 core and its C terminus. Thus, the acquisition of different (species-specific) C termini during evolution not only functionally separated cryptochromes from photolyase but also caused diversity within the cryptochrome family (Chaves, 2006).

Expression of Cryptochrome

The mammalian retina contains an endogenous circadian pacemaker that broadly regulates retinal physiology and function, yet the cellular origin and organization of the mammalian retinal circadian clock remains unclear. Circadian clock neurons generate daily rhythms via cell-autonomous autoregulatory clock gene networks. Thus, to localize circadian clock neurons within the mammalian retina, the cell type-specific expression of six core circadian clock genes was examined in individually identified mouse retinal neurons, and the clock gene expression rhythms in retinal degeneration (rd) mouse retinas were characterized. Individual photoreceptors, horizontal, bipolar, dopaminergic (DA) amacrines, catecholaminergic (CA) amacrines, and ganglion neurons were identified either by morphology or by a tyrosine hydroxylase (TH) promoter-driven red fluorescent protein (RFP) fluorescent reporter. Cells were collected, and their transcriptomes were subjected to multiplex single-cell RT-PCR for the core clock genes Period (Per) 1 and 2, Cryptochrome (Cry) 1 and 2, Clock, and Bmal1. Individual horizontal, bipolar, DA (dopaminergic), CA, and ganglion neurons, but not photoreceptors, were found to coordinately express all six core clock genes, with the lowest proportion of putative clock cells in photoreceptors (0%) and the highest proportion in DA neurons (30%). In addition, clock gene rhythms were found to persist for >25 days in isolated, cultured rd mouse retinas in which photoreceptors had degenerated. These results indicate that multiple types of retinal neurons are potential circadian clock neurons that express key elements of the circadian autoregulatory gene network and that the inner nuclear and ganglion cell layers of the mammalian retina contain functionally autonomous circadian clocks (Ruan, 2006).

Cryptochrome-Clock interaction

The molecular oscillator that drives circadian rhythmicity in mammals obtains its near 24-h periodicity from posttranslational regulation of clock proteins. Activity of the major clock kinase casein kinase I (CKI) epsilon is regulated by inhibitory autophosphorylation. Protein phosphatase (PP) 5 regulates the kinase activity of CKIepsilon. Cryptochrome regulates clock protein phosphorylation by modulating the effect of PP5 on CKIepsilon. Like CKIepsilon, PP5 is expressed both in the master circadian clock in the suprachiasmatic nuclei and in peripheral tissues independent of the clock. Expression of a dominant-negative PP5 mutant reduces Per phosphorylation by CKIepsilon in vivo, and down-regulation of PP5 significantly reduces the amplitude of circadian cycling in cultured human fibroblasts. Collectively, these findings indicate that PP5, CKIepsilon, and cryptochrome dynamically regulate the mammalian circadian clock (Partch, 2006).

The circadian clock is driven by cell-autonomous transcription/translation feedback loops. The BMAL1 transcription factor is an indispensable component of the positive arm of this molecular oscillator in mammals. A molecular genetic screening assay for mutant circadian clock proteins is presented that is based on real-time circadian rhythm monitoring in cultured fibroblasts. By using this assay, a domain was identified in the extreme C terminus of BMAL1 that plays an essential role in the rhythmic control of E-box-mediated circadian transcription. Remarkably, the last 43 aa of BMAL1 are required for transcriptional activation, as well as for association with the circadian transcriptional repressor Cryptochrome 1 (CRY1), depending on the coexistence of Clock protein. C-terminally truncated BMAL1 mutant proteins still associate with mPER2 (another protein of the negative feedback loop), suggesting that an additional repression mechanism may converge on the N terminus. Taken together, these results suggest that the C-terminal region of BMAL1 is involved in determining the balance between circadian transcriptional activation and suppression (Kiyohara, 2006).

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. A molecular genetic screen was developed in mammalian cells to identify mutants of the circadian transcriptional activators Clock and BMAL1, which were uncoupled from Cryptochrome-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of Clock and the C terminus of BMAL1 result in synergistic insensitivity through reduced physical interactions with Cry. Coexpression of these mutant proteins in cultured fibroblasts causes arrhythmic phenotypes in population and single-cell assays. These data demonstrate that Cry-mediated repression of the Clock/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function (Sato, 2006).

Timeless and Period interactions with Cryptochrome

The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. The human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein (Unsal-Kacmaz, 2005).

Receptor-mediated nucleocytoplasmic transport of clock proteins is an important, conserved element of the core mechanism for circadian rhythmicity. A systematic analysis of the nuclear export characteristics for the different murine period (mPER) and cryptochrome (mCRY) proteins using Xenopus oocytes as an experimental system demonstrates that all three mPER proteins, but neither mCRY1 nor mCRY2, are exported if injected individually. However, nuclear injection of heterodimeric complexes that contain combinations of mPER and mCRY proteins shows that mPER1 serves as an export adaptor for mCRY1 and mCRY2. Functional analysis of dominant-negative mPER1 variants designed either to sequester mPER3 to the cytoplasm or to inhibit nuclear export of mCRY1/2 in synchronized, stably transfected fibroblasts suggests that mPER1-mediated export of mCRY1/2 defines an important new element of the core clock machinery in vertebrates (Loop, 2005).

Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation

Period (PER) proteins are essential components of the mammalian circadian clock. They form complexes with cryptochromes (CRY), which negatively regulate CLOCK/BMAL1-dependent transactivation of clock and clock-controlled genes. To define the roles of mammalian CRY/PER complexes in the circadian clock, the crystal structure of a complex comprising the photolyase homology region of mouse CRY1 (mCRY1) and a C-terminal mouse PER2 (mPER2) fragment was determined. mPER2 winds around the helical mCRY1 domain covering the binding sites of FBXL3 and CLOCK/BMAL1, but not the FAD binding pocket. The structure revealed an unexpected zinc ion in one interface, which stabilizes mCRY1-mPER2 interactions in vivo. Evidence is provided that mCRY1/mPER2 complex formation is modulated by an interplay of zinc binding and mCRY1 disulfide bond formation, which may be influenced by the redox state of the cell. These studies may allow for the development of circadian and metabolic modulators (Schmalen, 2014).

A positive role for PERIOD in mammalian circadian gene expression

In the current model of the mammalian circadian clock, PERIOD (PER) represses the activity of the circadian transcription factors BMAL1 and CLOCK, either independently or together with CRYPTOCHROME (CRY). This study provides evidence that PER has an entirely different function from that reported previously, namely, that PER inhibits CRY-mediated transcriptional repression through interference with CRY recruitment into the BMAL1-CLOCK complex. This indirect positive function of PER is consistent with previous data from genetic analyses using Per-deficient or mutant mice. Overall, the results support the hypothesis that PER plays different roles in different circadian phases: an early phase in which it suppresses CRY activity, and a later phase in which it acts as a transcriptional repressor with CRY. This buffering effect of PER on CRY might help to prolong the period of rhythmic gene expression. Additional studies are required to carefully examine the promoter- and phase-specific roles of PER (Akashi, 2014).

Effects of mutation of mammalian cryptochrome

In the mouse, the core mechanism for the master circadian clock consists of interacting positive and negative transcription and translation feedback loops. Analysis of Clock/Clock mutant mice, homozygous Period2Brdm1 mutants, and Cryptochrome-deficient mice reveals substantially altered Bmal1 rhythms, consistent with the dominant role of Period2 in the positive regulation of the Bmal1 loop. In vitro analysis of Cryptochrome inhibition of Clock:BMAL1 heterodimer-mediated transcription shows that the inhibition is through direct protein:protein interactions, independent of the Period and Timeless proteins. Period2 is a positive regulator of the Bmal1 loop, and Cryptochromes are the negative regulators of the Period and Cryptochrome cycles (Shearman, 2000).

Bmal1 mRNA rhythm has been documented in mouse SCN using in situ hybridization with an antisense riboprobe to the two major Bmal1 transcripts found in the SCN. Wild-type mice exhibit a robust circadian rhythm in Bmal1 RNA levels, with low levels from circadian time (CT) 6 to 9 and peak levels from CT 15 to 18. The phase of the Bmal1 rhythm is opposite that of the mPer1, mPer2, and mPer3 RNA rhythms. In addition to driving rhythmic transcription of the mPer and mCry genes, it seems possible that CLOCK:BMAL1 heterodimers might simultaneously negatively regulate Bmal1 gene expression, as proposed for Clk regulation in Drosophila. If CLOCK:BMAL1 heterodimers negatively regulate Bmal1 gene expression and if the mutant CLOCK protein is ineffective in this activity, then Bmal1 RNA levels should be elevated and less rhythmic in homozygous Clock mutant (Clk/Clk) mice. Compared with wild types, however, Clk/Clk animals express a severely dampened circadian rhythm of Bmal1 RNA levels in the SCN. Trough Bmal1 RNA levels do not differ between Clk/Clk mice and wild types. The peak level of the RNA rhythm in homozygous Clk mutant mice is only ~30% of the peak value in wild types (Shearman, 2000).

The temporal profile of Clk RNA levels in the SCN of Clk/Clk mutant animals was examined, because Clk RNA levels have been shown to be decreased in the eye and hypothalamus of Clk/Clk mutant mice. Clk RNA levels do not manifest a circadian oscillation in mouse SCN. Clk RNA levels in the SCN of Clk/Clk mutant mice are not significantly different from those in the SCN of wild-type animals. Thus, the Clk mutation appears to alter regulation of Bmal1 gene expression in SCN, but not the regulation of the Clk gene itself. Clk expression may be decreased in other hypothalamic regions (Shearman, 2000).

The low levels of Bmal1 RNA in the SCN of homozygous Clk mutant animals show that CLOCK is not required for the negative regulation of Bmal1. Instead, these data indicate that CLOCK is actually necessary for the positive regulation of Bmal1. The positive effect of CLOCK on Bmal1 levels is probably indirect and may occur through the mPER and/or mCRY proteins, which are expressed in the nucleus of SCN neurons at the appropriate circadian time to enhance Bmal1 gene expression. In addition, the mPer1, mPer2, mPer3, mCry1, and mCry2 RNA oscillations are all down-regulated in Clk/Clk mutant mice. Reduced levels of the protein products of one or more of these genes may lead to the reduced levels of Bmal1 in the mutant mice, through loss of a positive drive on Bmal1 transcription (Shearman, 2000).

Homozygous mPer2Brdm1 mutant animals have depressed mPer1 and mPer2 RNA rhythms. The Bmal1 rhythm was examined in homozygous mPer2Brdm1 mutants to determine whether the positive drive on the Bmal1 feedback loop might come from the mPER2 protein. The effects of this mutation on the mCry1 RNA rhythm were also examined. The temporal profiles of gene expression were analyzed at six time points over the first day in DD in homozygous mPer2Brdm1 mutant mice and wild-type littermates. The Bmal1 RNA rhythm in the SCN of wild-type animals is substantially different from that of mutant mice. Trough RNA levels do not differ between wild-type and mutant animals, but the increase in Bmal1 RNA levels is advanced and truncated in the mutants, compared with the wild-type rhythm (Shearman, 2000).

The mCry1 RNA rhythm is also significantly altered. In the SCN of mPer2Brdm1 mutant mice, the peak levels of the mCry1 RNA rhythm are suppressed by ~50%, as reported for mPer1 and mPer2 RNA rhythms in this mouse line. These data suggest that maintenance of a normal Bmal1 RNA rhythm is important for the positive transcriptional regulation of the mPer and mCry feedback loops. Thus, rhythmic Bmal1 RNA levels may drive rhythmic BMAL1 levels, which, in turn, regulate CLOCK:BMAL1-mediated transcriptional enhancement in the master clock. Indeed, mPer1, mPer2, and mCry1 RNA rhythms are all blunted in the SCN of mPer2Brdm1 mutant mice, in which the Bmal1 rhythm is also blunted. In addition, the homozygous mPer2Brdm1 mutation is associated with a shortened circadian period and ensuing arrhythmicity in DD (Shearman, 2000).

These data, along with the fact that Clk RNA levels are unaltered in the SCN of homozygous mPer2Brdm1 mutants, also provide evidence that mPER2 is a positive regulator of the Bmal1 RNA rhythm. This effect may be unique to mPER2. For example, the diurnal oscillation in mPer2 RNA is not altered in the SCN of mPer1-deficient mice, and mPer1, mPer2, and Bmal1 RNA circadian rhythms are not altered in the SCN of mPer3-deficient mice. Moreover, circadian rhythms in behavior are sustained in mice deficient in either mPer1 or mPer3 (Shearman, 2000).

There are at least two ways that the mPer2Brdm1 mutation could alter the positive drive of the clock feedback loops. The mutation could disrupt mPER:mCRY interactions important for the synchronous oscillations of their nuclear localization and/or alter the protein's ability to interact with other proteins (e.g., transcription factors). Experiments show that functional mPER2:mCRY interactions are not mediated through the PAS domain. Similarly, the PAS domain is not important for the mCRY-mediated nuclear translocation of mPER1 in COS-7 cells (Shearman, 2000).

Because the mPER:mCRY interactions necessary for nuclear transport occur outside the PAS region, the PAS domain of an mPER2:mCRY heterodimer might be free to bind to an activator (e.g., transcription factor) and shuttle it into the nucleus to activate Bmal1 transcription. Alternatively, once in the nucleus, mPER2:mCRY heterodimers or mPER2 monomers could coactivate Bmal1 transcription through a PAS-mediated interaction with a transcription factor. mPER2 itself does not have a DNA-binding motif (Shearman, 2000).

The tonic mid-to-high mPer1 and mPer2 RNA levels in mCry-deficient mice suggest that CLOCK:BMAL1 heterodimers might be constantly driving mPer1 and mPer2 gene expression in the absence of transcriptional inhibition by the mCRY proteins. To examine whether Bmal1 RNA levels would also be modestly elevated, Bmal1 RNA levels in the SCN of mCry-deficient mice were compared with those in the SCN of wild-type mice of the same genetic background at CT 6 and at CT 18. Clk RNA levels were also examined in these animals. In wild-type animals, the typical circadian variation in Bmal1 RNA levels is apparent with high levels at CT 18 and low levels at CT 6. In contrast, inmCry-deficient mice, Bmal1 RNA levels are low at both circadian times. It is suggested that Clk RNA levels do not differ as a function of circadian time or genotype (Shearman, 2000).

The unexpected low Bmal1 gene expression in the SCN of mCry-deficient mice suggests that the Bmal1 feedback loop is disrupted in the mutant animals, with a resultant nonfunctional circadian clock. Nevertheless, enough Bmal1 gene expression and protein synthesis occurs for heterodimerization with CLOCK so that, without the strong negative feedback normally exerted by the mCRY proteins, mPer1 and mPer2 gene expression is driven sufficiently by the heterodimer to give intermediate to high RNA values (depending on RNA stability) (Shearman, 2000).

The mid-to-high mPer1 and mPer2 RNA levels in the SCN of mCry-deficient mice and simultaneous low Bmal1 levels suggest that mPER1 and mPER2 proteins may not be exerting much positive or negative influence on the core feedback loops. To test this, it had to be determined whether mPER1 and mPER2 are tonically expressed in the nuclei of SCN cells in mCry-deficient mice, because nuclear location appears necessary for action on transcription. mPER1 immunoreactivity exhibits a robust rhythm of nuclear staining in the SCN of wild-type mice, with high values at CT 12 and significantly lower values at CT 24 (Shearman, 2000). In mCry-deficient mice, however, mPER1 immunoreactivity is detected in the nucleus of a similar number of SCN neurons at each of the two circadian times (CT 12 and CT 24), and the values at each time were at ~40% of those seen at peak (CT 12) in wild-type animals (Shearman, 2000).

The double mCry mutation also alters the subcellular distribution of mPER1 staining in the SCN. In wild-type mice, mPER1 staining viewed under contrast interference is nuclear with a very condensed immunoreaction and a clear nucleolus. The neuropil of the SCN in wild types is devoid of mPER1 immunoreactivity. In the SCN of mCry-deficient animals, mPER1 staining is nuclear, but the nuclear profiles are less well defined and less intensely stained: perinuclear, cytoplasmic immunoreaction can be observed. In addition, the neuropil staining for mPER1 is higher in mCry-deficient mice, although dendritic profiles are not discernible. In the same brains, the constitutive nuclear staining for mPER1 normally seen in the piriform cortex is not altered in mCry-deficient animals (Shearman, 2000).

mPER2 immunoreactivity also exhibits a robust rhythm of nuclear staining in the SCN of wild-type mice, with high values at CT 12 and significantly lower counts at CT 24. In contrast, the pattern of mPER2 immunoreactivity in the SCN of mCry-deficient mice is markedly altered, with few mPER2 immunoreactive cells in the SCN of mCry-deficient animals at either circadian time (CT 12 or CT 24) (Shearman, 2000).

In the wild-type mice, the mPER2 staining profiles are nuclear, with well-defined outlines and nucleoli devoid of reaction product. In the few mPER2 immunoreactive cells in the SCN of mCry-deficient mice, low-level mPER2 staining is observed in the nucleus, but the profiles are poorly defined and low-intensity perinuclear staining can also be observed. As for mPER1, genotype has no discernible effect on nuclear mPER2 immunoreactivity in the piriform cortex, although there is evidence of a low level of perinuclear immunoreactivity for mPER2 in piriform cortex of mCry-deficient mice. The marked reduction of mPER2 staining in the SCN of mCry-deficient animals suggests that the mCRY proteins are either directly or indirectly important for mPER2 stability, because mPer2 RNA levels are at tonic intermediate to high levels in mCry-deficient mice, similar to those found for mPer1 RNA levels. The low levels of mPER2 immunoreactivity in the SCN of mCry-deficient mice, in conjunction with tonically low Bmal1 RNA levels, are consistent with an important role of mPER2 in the positive regulation of the Bmal1 loop. Because mPER1 is present in SCN nuclei in mCry-deficient mice, yet Bmal1 RNA is low, mPER1 likely has little effect on the positive regulation of the Bmal1 feedback loop or negative regulation of the mPer1, mPer2, and mPer3 cycles (Shearman, 2000).

mPER1 and mPER2 can each enter the nucleus even in the absence of mCRY:mPER interactions. mPER1 is expressed in the nucleus of SCN neurons from mCry-deficient mice, and both mPER1 and mPER2 are constitutively expressed in the nucleus of cells in the piriform cortex of mCry-deficient animals. The phosphorylation state of mPER1 dictates its cellular location in the absence of mPER1:mCRY interactions, because its phosphorylation by casein kinase I epsilon leads to cytoplasmic retention in vitro. Thus, the nuclear location of both mPER1 and mPER2 in vivo may depend on several factors, including interactions with mCRY and other proteins and their phosphorylation (Shearman, 2000).

The intermediate to high levels of mPer1 and mPer2 gene expression throughout the circadian day in mCry-deficient mice are consistent with a prominent role of the mCRY proteins in negatively regulating CLOCK:BMAL1-mediated transcription. When cotransfected, mouse (m)CLOCK and syrian hamster (sh)BMAL1 heterodimers induce a large increase in transcriptional activity (1744-fold) that is reduced by >90% by mCRY1 or mCRY2. The mCLOCK: shBMAL1- and hMOP4:shBMAL1-induced transcription in S2 cells is dependent on an intact CACGTG E box, because neither heterodimer causes an increase in transcription when a mutated E box reporter is used in the transcriptional assay. Immunofluorescence of epitope-tagged mCRY1 or mCRY2 expressed in S2 cells shows that each is >90% nuclear in location, as in mammalian cells (Shearman, 2000).

These data indicate that mCRY1 and mCRY2 are nuclear proteins that can each inhibit mCLOCK:shBMAL1-induced transcription independent of the mPER and mTIM proteins and of each other. The results also show that the inhibitory effect is not mediated by the interaction of either mCRY1 or mCRY2 with the E box itself, because E box-mediated transcription is not blocked by the mCRY proteins when transcription is activated by dCLOCK:CYC heterodimers. It thus appears that the mCRY proteins inhibit mCLOCK: shBMAL1-mediated transcription by interacting with either or both of the transcription factors, because a similar inhibition is found with hMOP4:shBMAL1-induced transcription. Yeast two-hybrid assays have revealed strong interactions of each mCRY protein with mCLOCK and shBMAL1. Weaker interactions have been detected between each mCRY protein and hMOP4. This is further evidence of functionally relevant associations of each mCRY protein with each of the three transcription factors. Attempts were made to determine whether the mCRY-induced inhibition of transcription is through interaction with CLOCK and/or BMAL1. mCRY inhibits mCLOCK:shBMAL1-induced transcription through interaction with either mCLOCK alone or through an association with both mCLOCK and BMAL1 in a multiprotein complex (Shearman, 2000).

A working model of the SCN clockwork proposes three types of interacting molecular loops. The mCry genes comprise one loop, with true autoregulatory, negative feedback, in which the protein products feed back to turn off their own transcription. The second loop is that manifested by each of the mPer genes and some clock controlled output genes (CCGs: for example, vasopressin prepropressophysin). This loop type is driven by the same positive elements (CLOCK:BMAL1) as the mCry loop, but transcription is not turned off by the respective gene products. Instead, the mCRY protein acts as a negative regulator, leaving the protein products free for other actions. Thus, mPER2 positively drives transcription of the Bmal1 gene, mPER1 may function to stabilize protein components of the loop, and CCG products (which might include mPER3) function as output signals. The rhythmic regulation of Bmal1 comprises the third loop with rhythmicity controlled by the cycling presence and absence of a positive element dependent on mPER2. This positive feedback loop augments the positive regulation of the first two loops (Shearman, 2000).

This model of interacting loops proposes that at the start of the circadian day, mPer and mCry transcription are driven by accumulating CLOCK:BMAL1 heterodimers acting through E box enhancers. After a delay, the mPER and mCRY proteins are synchronously expressed in the nucleus where the mCRY proteins shut off CLOCK:BMAL1-mediated transcription by directly interacting with these transcription factors. At the same time that the mCRY proteins are inhibiting CLOCK:BMAL1-mediated transcription, mPER2 either shuttles a transcriptional activator into the nucleus or coactivates a transcriptional complex to enhance Bmal1 transcription. The importance of the Bmal1 RNA rhythm is to drive a BMAL1 rhythm after a 4- to 6-hour delay. This delay in the protein rhythm would provide increasingly available CLOCK:BMAL1 heterodimers at the appropriate circadian time to drive mPer/mCry transcription, thereby restarting the cycle. It is thus predicted that BMAL1 availability is rate limiting for heterodimer formation and critical for restarting the loops. Delineating factors that regulate clock protein stability and interactions (phosphorylation and proteolysis) are important next steps for defining how a 24-hour time constant is built into the clockwork (Shearman, 2000).

The core oscillator driving the circadian clock is located in the ventral part of the hypothalamus, the so called suprachiasmatic nuclei (SCN). At the molecular level, this oscillator is thought to be composed of interlocking autoregulatory feedback loops involving a set of clock genes. Among the components driving the mammalian circadian clock are the Period 1 and 2 (mPer1 and mPer2) and Cryptochrome 1 and 2 (mCry1 and mCry2) genes. A mutation in the mPer2 gene leads to a gradual loss of circadian rhythmicity in mice kept in constant darkness (DD). Inactivation of the mCry2 gene in mPer2 mutant mice restores circadian rhythmicity and normal clock gene expression patterns. Thus, mCry2 can act as a nonallelic suppressor of mPer2, which points to direct or indirect interactions of PER2 and CRY2 proteins. In marked contrast, inactivation of mCry1 in mPer2 mutant mice does not restore circadian rhythmicity but instead results in complete behavioral arrhythmicity in DD, indicating different effects of mCry1 and mCry2 in the clock mechanism (Oster, 2002).

The mPer1, mPer2, mCry1, and mCry2 genes play a central role in the molecular mechanism driving the central pacemaker of the mammalian circadian clock, located in the suprachiasmatic nuclei (SCN) of the hypothalamus. In vitro studies suggest a close interaction of all mPER and mCRY proteins. mPER and mCRY interactions in vivo were investigated by generating different combinations of mPer/mCry double-mutant mice. mCry2 acts as a nonallelic suppressor of mPer2 in the core clock mechanism. Focus was placed on the circadian phenotypes of mPer1/mCry double-mutant animals; a decay of the clock with age was found in mPer1-/- mCry2-/- mice at the behavioral and the molecular levels. These findings indicate that complexes consisting of different combinations of mPER and mCRY proteins are not redundant in vivo and have different potentials in transcriptional regulation in the system of autoregulatory feedback loops driving the circadian clock (Oster, 2003).

Old mPer1-/- mCry2-/- mice synchronize poorly to the light dark cycle. Therefore, tests were performed to see whether CREB, an essential factor for numerous transcriptional processes, is activated by phosphorylation in response to a light pulse. CREB phosphorylation was only slightly lowered in young mPer1-/- mCry2-/- mice but was significantly impaired in old animals, indicating a defect in light signaling in the SCN of these mice. At the behavioral level, the phase shifts of only young mPer1-/- mCry2-/- mice could be measured, because old animals immediately became arrhythmic in DD. The young mPer1-/- mCry2-/- mice resemble mPer1-/- animals in that they are not able to advance clock phase, suggesting that this anomaly is due to the absence of mPer1 (Oster, 2003).

The impaired light response of mPer1-/- mCry2-/- mice might be a consequence of a defect in transmitting light information from the eye to the SCN. To test this possibility, anatomical malformations in the retina were sought. Neither young nor old mPer1-/- mCry2-/- mice displayed overt abnormalities in retinal morphology. Comparable to the SCN, however, light-dependent phosphorylation of CREB at Ser 133 was affected in old mPer1-/- mCry2-/- mice. As a consequence, light perceived by the eye is probably not processed properly to induce cellular signaling. The reason for the impaired transmission of the light signal is most likely not a developmental defect, because young mPer1-/- mCry2-/- mice show phosphorylation of CREB at Ser 133. Therefore, the defect is probably of transcriptional or posttranscriptional nature. The lack of phosphorylation of CREB might lead to an altered expression of melanopsin in ganglion cells. These cells are probably responsible for resetting of the clock by light. Hence, a reduced expression of melanopsin would affect resetting. This is in line with the recent finding, that melanopsin-deficient mice display attenuated clock resetting in response to brief light pulses, similar to what is observed in mPer1-/- mCry2-/- mice. In old mPer1-/- mCry2-/- mice, this might even lead to the poor synchronization of these mice to the LD cycle. Future studies will reveal whether melanopsin expression in ganglion cells of the retina is affected in old mPer1-/- mCry2-/- mice (Oster, 2003).

Circadian time-place learning in mice depends on Cry genes

Endogenous biological clocks allow organisms to anticipate daily environmental cycles. The ability to achieve time-place associations is key to the survival and reproductive success of animals. The ability to link the location of a stimulus (usually food) with time of day has been coined time-place learning, but its circadian nature was only shown in honeybees and birds. So far, an unambiguous circadian time-place-learning paradigm for mammals is lacking. This study analyzed whether expression of the clock gene Cryptochrome (Cry), crucial for circadian timing, is a prerequisite for time-place learning. Time-place learning in mice was achieved by developing a novel paradigm in which food reward at specific times of day was counterbalanced by the penalty of receiving a mild footshock. Mice lacking the core clock genes Cry1 and Cry2 (Cry double knockout mice; Cry1-/-Cry2-/-) learned to avoid unpleasant sensory experiences (mild footshock) and could locate a food reward in a spatial learning task (place preference). These mice failed, however, to learn time-place associations. This specific learning and memory deficit shows that a Cry-gene dependent circadian timing system underlies the utilization of time of day information. These results reveal a new functional role of the mammalian circadian timing system (Van der Zee, 2008).

Most likely, Cry1-/-Cry2-/- mice failed to master the time-place task as a consequence of lacking an intact circadian system. Hence, acquisition and transmission of time-of-day information (either directly or indirectly via Cry genes) to brain regions and mechanisms underlying the formation of time-place associations is hampered. This implies that various times of day cannot be distinguished, rendering it impossible to form time-place associations. Alternatively, but more unlikely, these mice may fail to associate different times of day with different spatial locations as a consequence of the loss of Cry genes. It could also be argued that Cry1-/-Cry2-/- mice failed this task in contrast to the other learning tasks because it is more difficult to achieve. The rate of acquisition for time-place learning and Y maze spatial learning are, however, comparable. This renders it unlikely that the failure of the Cry1-/-Cry2-/- mice to perform time-place learning is due to a higher cognitive demand of this task, although the data cannot completely rule out the possibility that Cry1-/-Cry2-/- mice have a more general deficit to perform three-way conditional spatial discriminations (not just those in which time of day acts as the conditional cue). No differences in overt behavior were observed in the various steps of the experimental procedure, and the Cry1-/-Cry2-/- mice could obviously smell and locate a food reward and were responsive to the applied footshocks. Taken together, it is considered unlikely that other (minor and more generalized) deficits in Cry1-/-Cry2-/- mice not related to the circadian system are responsible for the specific failure of performing time-place learning (Van der Zee, 2008).

Crytochrome degradation

Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557-phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by approximately 4 h in both light-dark cycle and constant dark conditions. The Ser-557-phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3beta (GSK-3beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3beta at Ser-9, which negatively regulates the kinase activity, GSK-3beta exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein (Harada, 2005).

One component of the circadian clock in mammals is the Clock-Bmal1 heterodimeric transcription factor. Among its downstream targets, two genes, Cry1 and Cry2, encode inhibitors of the Clock-Bmal1 complex that establish a negative-feedback loop. Both Cry1 and Cry2 proteins are ubiquitinated and degraded via the SCFFbxl3 ubiquitin ligase complex. This regulation by SCFFbxl3 is a prerequisite for the efficient and timely reactivation of Clock-Bmal1 and the consequent expression of Per1 and Per2, two regulators of the circadian clock that display tumor suppressor activity. Silencing of Fbxl3 produced no effect in Cry1–/–;Cry2–/– cells, which shows that Fbxl3 controls clock oscillations by mediating the degradation of CRY proteins (Busino, 2007).

Using a forward genetics ENU mutagenesis screen for recessive mutations that affect circadian rhythmicity in the mouse, a long period (~26 hr) circadian mutant was isolated named Overtime (Ovtm). Positional cloning and genetic complementation reveal that Ovtm encodes the the F-box protein FBXL3, a component of the SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligase complex. The Ovtm mutation causes an isoleucine to threonine (I364T) substitution leading to a loss of function in FBXL3, which interacts specifically with the Cryptochrome (Cry) proteins. In Ovtm mice, expression of the Period proteins PER1 and PER2 is reduced; however, the Cry proteins Cry1 and Cry2 are unchanged. The loss of FBXL3 function leads to a stabilization of the Cry proteins, which in turn leads to a global transcriptional repression of the Per and Cry genes. Thus, Fbxl3Ovtm defines a molecular link between CRY turnover and CLOCK/BMAL1-dependent circadian transcription to modulate circadian period (Siepka, 200


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