cryptochrome

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).

Effects of Mutation or Deletion

A new rhythm mutation, cry^b, 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, cry^b is an apparent null mutation in a gene encoding Drosophila's version of the blue light receptor cryptochrome. Behaviorally, cry^b 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. cry^b flies are rhythmic in constant darkness, correlating with robust Per and Tim cycling in certain pacemaker neurons (Stanewsky, 1998).

The cry^b 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 cry^b flies are subjected to light pulses, no clear phase shifts result. This seems to contradict the fact that cry^b 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 cry^b 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 cry^b 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 cry^b mutant's eyes. The LNs consist of two groups of cells in each side of the brain, ~6 neurons in a relatively dorsal cluster (LN_ds) and ~10 such cells in a more ventrally located one (LN_vs). Clock functions in the LN_vs (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 cry^b 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, LN_ds, and LN_vs) were scored blindly. Both proteins are observed to cycle in the LNs of cry^b 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 cry^b 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, cry^b, 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 cry^b flies are still able to reset their circadian rhythm (entrain) to new light-dark cycles. cry^b 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 cry^b 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 cry^b flies. In intense constant light, the cry^b 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 cry^b mutant strain under constant light conditions. The free-running, circadian nature of cry^b flies' constant-light behavior is further demonstrated by the 19.2-h period observed for flies with cry^b in combination with a short allele of the period gene (per ^s) (Emery, 2000a).

These results show that the cry^b 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 cry^b flies entrain to different 24-h light-dark cycles? The missense cry^b 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 cry^b 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 cry^b 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, cry^b, inhibits circadian photoresponses. Restricting Cry expression to specific fly tissues shows that Cry expression is needed in a cell-autonomous fashion for oscillators present in different locations. Cry overexpression in brain pacemaker cells increases behavioral photosensitivity, and this restricted Cry expression also rescues all circadian defects of cry^b behavior. As wild-type pacemaker neurons express Cry, the results indicate that they make a striking contribution to all aspects of behavioral circadian rhythms and are directly light responsive. These brain neurons therefore contain an identified deep brain photoreceptor, as well as the other circadian elements: a central pacemaker and a behavioral output system (Emery, 2000b).

The mutant cry^b 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 cry^b 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; cry^b 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 Cry^B overexpression and the absence of Cry^B abundance cycling under LD conditions, cry^b is probably a strong hypomorphic allele that encodes a protein without photoreceptor activity. This conclusion is consistent with the results of Cry^B expression studies in heterologous systems (Emery, 2000b and references therein).

Behavioral rescue experiments show that all known photoresponse defects of cry^b 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 per^S; cry^b and norpA^P41; cry^b 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 cry^b, 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 (LN_v), 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;gl^60j), and retaining the adult eyelet but lacking compound eyes as well as Cryptochrome (so¹;cry^b). 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 glass^60j;cry^b 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, itís 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 rhythmís 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 norpA^P41;cry^b 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 norpA^P41;cry^b 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 gl^60j;cry^b combination revealed in this study. Most norpA^P41;cry^b individuals are still able to entrain to LD cycles (Helfrich-Forster, 2001).

There are at least two possible explanations for these findings: cry^b 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 flyís 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 gl^60j 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 gl^60j;cry^b 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 cry^b; (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 gl^60j;cry^b, 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 gl^60j;cry^b 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, cry^b, and per⁰¹; cry^b 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; cry^b, 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 cry^b flies, splicing levels are significantly elevated, particularly after lights off. This is also the case when per⁰¹; cry^b is compared to WT. At 29°C, splicing levels are generally 5%-10% higher in per⁰¹, tim⁰¹, and cry^b 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; cry^b 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 cry^b. 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 gl^60j and norpA^P41 (Collins, 2004).

The proportion of per mRNA transcripts that are spliced at both temperatures is increased in both the norpA^P41 and gl^60j backgrounds compared to WT. At 18°C, ~65% of per transcripts are spliced in norpA^P41 and ~60% in gl^60j, whereas at 29°C, these levels fall to ~55% and ~40%, respectively. Apart from a marginal difference between norpA^P41; cry^b, and norpA^P41 at 18°C, there are no significant effects for either norpA^P41 or gl^60j when combined with cry^b. 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 norpA^P41 or gl^60j (Collins, 2004).

Interestingly, in gl^60j, there is a 20% difference between the splicing levels at different temperatures (60%-40%), whereas in norpA^P41, this difference is reduced to 10% (65%-55%). The difference in gl^60j is similar to that seen in WT (45%-25%). Thus per splicing in norpA^P41 is relatively insensitive to temperature changes. It is also clear that the level of splicing in norpA^P41 is significantly higher at all times and temperatures than gl^60j. Therefore, the effect of norpA^P41 on splicing is greater than that of gl^60j_, despite gl^60j 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, cry^b and norpA^P41, but not for norpA^P41; cry^b or gl^60j, where despite elevated splicing levels at higher temperatures, there is no difference in the phase of activity. gl^60j cry^b 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 cry^b (50%) to gl^60j (60%) to norpA^P41 (65%), and at 29°C from WT (25%) to gl^60j and cry^b (~35%) to norpA^P41 (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 norpA^P41, cry^b, and WT, there is an inverse relationship between average splicing levels and the position of the activity peak at 18°C, with norpA^P41 and cry^b having similarly advanced activity peaks compared to WT. At 29°C, the same inverse relationship holds, with norpA^P41 advanced compared to cry^b, 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, norpA^P41; cry^b and gl^60j,, 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 gl^60j and norpA^P41; cry^b. Thus the per RNA profiles of gl^60j and norpA^P41 were compared to WT. Because per does not cycle in cry^b whole head homogenates, the underlying cycle in this background was not examined. WT and norpA^P41 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 gl^60j at either temperature, and levels of per are significantly different from WT and norpA^P41 (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 cry^b 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; cry^b 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, cry^b, and per⁰¹; cry^b. 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 cry^b 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 cry^b 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 cry^b 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 cry^b 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 cry^b all show the same splicing phenotype. However, if this splicing phenotype is simply what happens when the clock stops, then per⁰¹; cry^b should show the same splicing phenotype as either single mutant. This is not the case, because the daytime splicing in per⁰¹; cry^b at 29°C is dramatically elevated compared to either single mutant. Thus the splicing phenotypes of per⁰¹, tim⁰¹, and cry^b 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 gl^60j, there is no per mRNA cycle in whole head homogenates. This means that in the majority of cells in the gl^60j head, the clock has either stopped or cells have become desynchronized. If the former is true, then splicing levels of gl^60j 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).

norpA^P41 and gl^60j have considerably higher splicing levels than WT and cry^b 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 gl^60j cry^b and norpA^P41; cry^b double mutants. The splicing levels of gl^60j and gl^60j cry^b are similar at both temperatures, which is also true of norpA and norpA^P41; cry^b at 29°C. At 18°C, there is slightly more spliced per RNA in norpA^P41; cry^b than in the norpA^P41 single mutant, reflecting the earlier result where cry^b 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 cry^b, 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 cry^b mutants, there is no day-night difference in splicing levels at 29°C in either gl^60j or norpA^P41. 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 norpA^P41 and gl^60j 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, gl^60j, or norpA^P41 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 cry^b mutants are able to adapt the timing of locomotor activity to long and short photoperiods, whereas flies with defective visual photoreception, including gl^60j, are not (Collins, 2004).

Interestingly, although gl^60j is the more severe visual mutant, norpA^P41 has significantly higher per splicing levels than gl^60j 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 norpA^P41 (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 norpA^P41 fly's locomotor patterns seemingly more sensitive to high temperatures than WT. Additionally, norpA^P41 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 norpA^P41 mutants behave as if they have an impaired ability to detect high temperatures. norpA^P41 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 norpA^P41 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 norpA^P41 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, norpA^P41, and cry^b, 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 cry^b than in WT, resulting in the earlier evening activity peak seen in cry^b 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 norpA^P41: 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 cry^b (55% and 40%) and relates to the earlier phases of locomotor activity seen in norpA^P41 compared to the other genotypes. However, at 18°C there is more spliced per in norpA^P41 than in cry^b, 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 norpA^P41; cry^b and gl^60j, 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 gl^60j, norpA^P41, and WT flies were analyzed at 18°C and 29°C, it was found that whereas per levels cycle in norpA^p41 and WT, this cycle is lost in gl^60j. 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 gl^60j 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 gl^60j, 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 norpA^P41; cry^b double mutants, because in this case per mRNA is assumed to be noncycling because of the cry^b background. However, the locomotor behavior of cry^b 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 norpA^P41; cry^b) 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; cry^b 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 gl^60j cry^b or norpA^P41; cry^b, 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 cry^b 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 cry^b, 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; cry^b 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; cry^b oscillator is particularly informative because per⁰¹ is effectively rescuing this phenotype in cry^b. This can be understood in terms of the robust, high-amplitude oscillator in cry^b, being less 'perturbable' by light as Cry photoreception is lost, whereas the damped oscillator in per⁰¹; cry^b is more sensitive to the environmental stimulus, precisely because of its low amplitude. The damped oscillation in the per⁰¹; cry^b 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 cry^b 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 (LN_vs and LN_ds) 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 (sLN_vs) and dorsal LNs (LN_ds) 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-LN_vs) 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-LN_vs, than in the s-LN_vs or the LN_ds under LD conditions, suggesting that rhythmic output from the l-LN_vs are compromised in a cry^b background. In turn, this may contribute to the peculiar defects of cry^b 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-LN_v and LN_d oscillators in cry^b 'resist' the effects of brief light pulses, because of the impaired light input that is relayed to the s-LN_vs, and from the s-LN_vs to the LN_ds, by the more light-relevant l-LN_vs. In per⁰¹, the molecular clock is severely dampened in all clock neurons, more so in the s-LN_vs and LN_ds that have an endogenous cycle than the l-LN_vs that do not. Thus, the light-mediated input from the l-LN_v neurons into the s-LN_vs, and indirectly to the LN_ds, 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 cry^b and per⁰¹ are combined, the weak oscillator of per⁰¹ is no longer overcome by the light input because it is attenuated by cry^b and mediated via the l-LN_vs. 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-LN_vs 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-LN_vs to entrain the per⁰¹; cry^b double mutant so effectively, unless it is transmitted from another neuronal source: the l-LN_vs (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-LN_vs, suggesting that the l-LN_vs 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-LN_vs likely extend their dendritic arborizations..Although not definitive, these results support earlier claims that the photoreceptors may directly (or indirectly) synapse with the l-LN_vs. As stated above, these molecular and proposed functional differences between s- and l-LN_vs may also contribute to explaining the loss of light responsiveness in cry^b 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; cry^b, 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 LN_vs or LN_ds of per⁰¹; cry^b, 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; cry^b. 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).

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 cry^b mutants in LL (Picot, 2007).

The strong rhythmicity of the cry^b 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 cry^b to 22–23 h in cry^b pdf⁰ flies. An attractive possibility is that the strong short-period rhythm observed in the cry^b 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, our 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).

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date revised: 1 June 2008

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