Transcriptional feedback loop regulation, function, and ontogeny in Drosophila.

The Drosophila circadian oscillator is composed of interlocked period/timeless (per/tim) and Clock (Clk) transcriptional feedback loops. These feedback loops drive rhythmic transcription having peaks at dawn and dusk during the daily cycle and function in the brain and a variety of peripheral tissues. To understand how the circadian oscillator keeps time and controls metabolic, physiological, and behavioral rhythms, we must determine how these feedback loops regulate rhythmic transcription, determine the relative importance of the per/tim and Clk feedback loops with regard to circadian oscillator function, and determine how these feedback loops come to be expressed in only certain tissues. Substantial insight into each of these issues has been gained from experiments performed in our lab and others and is summarized here.

A new role for cryptochrome in a Drosophila circadian oscillator.

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. Here we show that 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.

Defining the role of Drosophila lateral neurons in the control of circadian rhythms in motor activity and eclosion by targeted genetic ablation and PERIOD protein overexpression.

The ventral lateral neurons (LNvs) of the Drosophila brain that express the period (per) and pigment dispersing factor (pdf) genes play a major role in the control of circadian activity rhythms. A new P-gal4 enhancer trap line is described that is mostly expressed in the LNvs This P-gal4 line was used to ablate the LNvs by using the pro-apoptosis gene bax, to stop PER protein oscillations by overexpressing per and to block synaptic transmission with the tetanus toxin light chain (TeTxLC). Genetic ablation of these clock cells leads to the loss of robust 24-h activity rhythms and reveals a phase advance in light-dark conditions as well as a weak short-period rhythm in constant darkness. This behavioural phenotype is similar to that described for disconnected1 (disco1) mutants, in which we show that the majority of the individuals have a reduced number of dorsally projecting lateral neurons which, however, fail to express PER. In both LNv-ablated and disco1 flies, PER cycles in the so-called dorsal neurons (DNs) of the superior protocerebrum, suggesting that the weak short-period rhythm could stem from these PDF-negative cells. The overexpression of per in LNs suppresses PER protein oscillations and leads to the disruption of both activity and eclosion rhythms, indicating that PER cycling in these cells is required for both of these rhythmic behaviours. Interestingly, flies overexpressing PER in the LNs do not show any weak short-period rhythms, although PER cycles in at least a fraction of the DNs, suggesting a dominant role of the LNs on the behavioural rhythms. Expression of TeTxLC in the LNvs does not impair activity rhythms, which indicates that the PDF-expressing neurons do not use synaptobrevin-dependent transmission to control these rhythms.

Survey of transcripts in the adult Drosophila brain.

BACKGROUND: Classic methods of identifying genes involved in neural function include the laborious process of behavioral screening of mutagenized flies and then rescreening candidate lines for pleiotropic effects due to developmental defects. To accelerate the molecular analysis of brain function in Drosophila we constructed a cDNA library exclusively from adult brains. Our goal was to begin to develop a catalog of transcripts expressed in the brain. These transcripts are expected to contain a higher proportion of clones that are involved in neuronal function. RESULTS: The library contains approximately 6.75 million independent clones. From our initial characterization of 271 randomly chosen clones, we expect that approximately 11% of the clones in this library will identify transcribed sequences not found in expressed sequence tag databases. Furthermore, 15% of these 271 clones are not among the 13,601 predicted Drosophila genes. CONCLUSIONS: Our analysis of this unique Drosophila brain library suggests that the number of genes may be underestimated in this organism. This work complements the Drosophila genome project by providing information that facilitates more complete annotation of the genomic sequence. This library should be a useful resource that will help in determining how basic brain functions operate at the molecular level.

The period E-box is sufficient to drive circadian oscillation of transcription in vivo.

The minimum element from the Drosophila period promoter capable of driving in vivo cycling mRNA is the 69 bp circadian regulatory sequence (CRS). In cell culture, an 18 bp E-box element from the period promoter is regulated by five genes that are involved in the regulation of circadian expression in flies. This E-box is a target for transcriptional activation by bHLH-PAS proteins dCLOCK (dCLK) and CYCLE (CYC), this activation is inhibited by PERIOD (PER) and TIMELESS (TIM) together, and inhibition of dCLK/CYC by PER and TIM is blocked by CRYPTOCHROME (CRY) in the presence of light. Here, the same 18 bp E-box region generated rhythmic expression of luciferase in flies under both light-dark cycling and constant conditions. Flies heterozygous for the Clke(jrk) mutation maintained rhythmic expression from the E-box although at a lower level than wild type. Homozygous mutant Clk(jrk) animals had drastically lowered and arrhythmic expression. In a per01 background, expression from the E-box was high and not rhythmic. Transcription mediated by the per E-box was restricted to the same spatial pattern as the CRS. The per E-box DNA element and cognate binding proteins can confer per-like temporal and spatial expression. This demonstrates in vivo that the known circadian genes that form the core of the circadian oscillator in Drosophila integrate their activities at a single DNA element.

Specific sequences outside the E-box are required for proper per expression and behavioral rescue.

A 69 bp circadian regulatory sequence (CRS) upstream of the per gene is sufficient to drive circadian transcription, mediate proper spatial expression, and rescue behavioral rhythmicity in per01 flies. Within the CRS, an E-box is required for transcriptional activation by two basic-helix-loop-helix (bHLH) PERARNT-SIM (PAS) transcription factors, dCLOCK (dCLK) and CYCLE (CYC). To define sequences within the CRS that are required for spatial expression, circadian expression, and behavioral rhythmicity, a series of mutants that alter blocks of 3 to 12 nucleotides across the entire CRS were used to drive lacZ or per expression in vivo. As expected, the E-box within the CRS is necessary for high-level expression and behavioral rhythmicity, but sequences outside the E-box are also required for transcriptional activation, proper spatial expression, and behavioral rhythmicity. These results indicate that the dCLK-CYC target site extends beyond the E-box and that factors other than dCLK and CYC modulate per transcription.

Disruption of synaptic transmission or clock-gene-product oscillations in circadian pacemaker cells of Drosophila cause abnormal behavioral rhythms.

To study the function of clock-gene-expressing neurons, the tetanus-toxin light chain (TeTxLC), which blocks chemical synaptic transmission, was expressed under the control of promoters of the clock genes period (per) and timeless (tim), each fused to GAL4-encoding sequences. Although TeTxLC did not affect cycling of a clock-gene product at the gross level, it disrupted the rhythmic behavior of adult Drosophila. In constant darkness, the proportion of rhythmic flies was reduced in flies expressing active TeTxLC compared to controls, including those expressing inactive toxin. The behavior of TeTxLC-expressing flies was less synchronized to light:dark cycles than that of controls. To determine which neurons are responsible for these effects on behavior, the toxin was also expressed in restricted subsets of per/tim-expressing, laterally located pacemaker neurons by expressing TeTxLC under the control of a driver in which GAL4-encoding sequences are fused to the promoter of the pigment dispersing factor (pdf) gene. pdf-gal4-driven TeTxLC expression had relatively little effect on behavioral rhythms, implying that per/tim neurons other than pdf-expressing lateral neurons participate in the generation of rhythmic behavior. In another set of experiments, period gene products were expressed under the control of per-gal4 or tim-gal4. This resulted in an increased level of PER protein in many brain cells and reduction of bioluminescence cycling reported by a per-luciferase transgene, especially in the case of per expression affected by tim-gal4. This indicates a disruption of the transcriptional feedback loop that is a part of the oscillatory mechanism underlying Drosophila's circadian rhythms. Consistent with this molecular defect, the proportion of rhythmic individuals in constant darkness was subnormal in flies expressing PER under the control of tim-gal4, and their behavior in light:dark cycles was abnormal.

dCLOCK is present in limiting amounts and likely mediates daily interactions between the dCLOCK-CYC transcription factor and the PER-TIM complex.

In Drosophila melanogaster four circadian clock proteins termed PERIOD (PER), TIMELESS (TIM), dCLOCK (dCLK), and CYCLE (CYC/dBMAL1) function in a transcriptional feedback loop that is a core element of the oscillator mechanism. dCLK and CYC are members of the basic helix-loop-helix (bHLH)/PAS (PER-ARNT-SIM) superfamily of transcription factors and are required for high-level expression of per and tim and repression of dClk, whereas PER and TIM inhibit dCLK-CYC-mediated transcription and lead to the activation of dClk. To understand further the dynamic regulation within the circadian oscillator mechanism, we biochemically characterized in vivo-produced CYC, determined the interactions of the four clock proteins, and calculated their absolute levels as a function of time. Our results indicate that throughout a daily cycle the majority of the dCLK present in adult heads stably interacts with CYC, indicating that CYC is the primary in vivo partner of dCLK. dCLK-CYC dimers are bound by PER and TIM during the late evening and early morning, suggesting the formation of a tetrameric complex with impaired transcriptional activity. Although dCLK is present in limiting amounts and CYC is by far the most abundant of the four clock proteins that have been examined, PER and TIM appear to interact preferentially with dCLK. Our results suggest that dCLK is the main component regulating the daily abundance of transcriptionally active dCLK-CYC complexes.

From biological clock to biological rhythms.

The genetic and molecular analysis of circadian timekeeping mechanisms has accelerated as a result of the increasing volume of genomic markers and nucleotide sequence information. Completion of whole genome sequences and the use of differential gene expression technology will hasten the discovery of the clock output pathways that control diverse rhythmic phenomena.

Interlocked feedback loops within the Drosophila circadian oscillator.

Drosophila Clock (dClk) is rhythmically expressed, with peaks in mRNA and protein (dCLK) abundance early in the morning. dClk mRNA cycling is shown here to be regulated by PERIOD-TIMELESS (PER-TIM)-mediated release of dCLK- and CYCLE (CYC)-dependent repression. Lack of both PER-TIM derepression and dCLK-CYC repression results in high levels of dClk mRNA, which implies that a separate dClk activator is present. These results demonstrate that the Drosophila circadian feedback loop is composed of two interlocked negative feedback loops: a per-tim loop, which is activated by dCLK-CYC and repressed by PER-TIM, and a dClk loop, which is repressed by dCLK-CYC and derepressed by PER-TIM.

Circadian rhythms in olfactory responses of Drosophila melanogaster.

The core mechanism of circadian timekeeping in arthropods and vertebrates consists of feedback loops involving several clock genes, including period (per) and timeless (tim). In the fruitfly Drosophila, circadian oscillations in per expression occur in chemosensory cells of the antennae, even when the antennae are excised and maintained in isolated organ culture. Here we demonstrate a robust circadian rhythm in Drosophila in electrophysiological responses to two classes of olfactory stimuli. These rhythms are observed in wild-type flies during light-dark cycles and in constant darkness, but are abolished in per or tim null-mutant flies (per01 and tim01) which lack rhythms in adult emergence and locomotor behaviour. Olfactory rhythms are also abolished in the per 7.2:2 transgenic line in which per expression is restricted to the lateral neurons of the optic lobe. Because per 7.2:2 flies do not express per in peripheral oscillators, our results provide evidence that peripheral circadian oscillators are necessary for circadian rhythms in olfactory responses. As olfaction is essential for food acquisition, social interactions and predator avoidance in many animals, circadian regulation of olfactory systems could have profound effects on the behaviour of organisms that rely on this sensory modality.

How a circadian clock adapts to seasonal decreases in temperature and day length.

We show that a thermosensitive splicing event in the 3' untranslated region of the mRNA from the period (per) gene plays an important role in how a circadian clock in Drosophila adapts to seasonally cold days (low temperatures and short day lengths). The enhanced splicing of this intron at low temperatures advances the steady state phases of the per mRNA and protein cycles, events that significantly contribute to the preferential daytime activity of flies on cold days. Because the accumulation of PER is also dependent on the photosensitive TIMELESS (TIM) protein, long photoperiods partially counteract the cold-induced advances in the oscillatory mechanism by delaying the daily increases in the levels of TIM. Our findings also indicate that there is a temperature-dependent switch in the molecular logic governing cycles in per mRNA levels.

Activating inhibitors and inhibiting activators: a day in the life of a fly.

The circadian clock keeps time through an intracellular oscillator that requires rhythmic gene expression. In Drosophila melanogaster, the core of this oscillator is composed of a circadian feedback loop in which the transcription of the period and timeless genes is repressed by their own protein products. In the past year, our understanding of clock organization and function in Drosophila has been advanced by breakthroughs that define when, where and how this feedback loop operates. These studies, along with those in other organisms, suggest that circadian feedback loops are widespread and that genes within these feedback loops are conserved between Drosophila and mammals.

Two alternatively spliced transcripts from the Drosophila period gene rescue rhythms having different molecular and behavioral characteristics.

The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The per gene is thought to encode three transcripts via differential splicing (types A, B, and C) that give rise to three proteins. Since the three per mRNA types were based on the analysis of cDNA clones, we tested whether these mRNA types were present in vivo by RNase protection assays and reverse transcriptase-mediated PCR. The results show that per generates two transcript types that differ only by the presence (type A) or absence (type B') of an alternative intron in the 3' untranslated region. Transgenic flies containing transgenes that produce only type B' transcripts (perB'), type A transcripts (perA), or both transcripts (perG) rescue locomotor activity rhythms with average periods of 24.7, 25.4, and 24.4 h, respectively. Although no appreciable differences in type A and type B' mRNA cycling were observed, a slower accumulation of PER in flies making only type A transcripts suggests that the intron affects the translation of per mRNA.

Drosophila photoreceptors contain an autonomous circadian oscillator that can function without period mRNA cycling.

Circadian oscillations in period (per) mRNA and per protein (PER) constitute, in part, a feedback loop that is required for circadian pacemaker function in Drosophila melanogaster. Oscillations in PER are required for oscillations in per mRNA, but the converse has not been rigorously tested because of a lack of measurable quantities of per mRNA and protein in the same cells. This circadian feedback loop operates synchronously in many neuronal and non-neuronal tissues, including a set of lateral brain neurons (LNs) that mediate rhythms in locomotor activity, but whether a hierarchy among these tissues maintains this synchrony is not known. To determine whether per mRNA cycling is necessary for PER cycling and whether cyclic per gene expression is tissue autonomous, we have generated per01 flies carrying a transgene that constitutively expresses per mRNA specifically in photoreceptors, a cell type that supports feedback loop function. These transformants were tested for different aspects of feedback loop function including per mRNA cycling, PER cycling, and PER nuclear localization. Under both light/dark (LD) cycling and constant dark (DD) conditions, PER abundance cycles in the absence of circadian cycling of per mRNA. These results show that per mRNA cycling is not required for PER cycling and indicate that Drosophila photoreceptors R1-R6 contain a tissue autonomous circadian oscillator.

A circadian enhancer mediates PER-dependent mRNA cycling in Drosophila melanogaster.

Genes expressed under circadian-clock control are found in organisms ranging from prokaryotes to humans. In Drosophila melanogaster, the period (per) gene, which is required for clock function, is transcribed in a circadian manner. We have identified a circadian transcriptional enhancer within a 69-bp DNA fragment upstream of the per gene. This enhancer drives high-amplitude mRNA cycling under light-dark-cycling or constant-dark conditions, and this activity is per protein (PER) dependent. An E-box sequence within this 69-bp fragment is necessary for high-level expression, but not for rhythmic expression, indicating that PER mediates circadian transcription through other sequences in this fragment.

per mRNA cycling is locked to lights-off under photoperiodic conditions that support circadian feedback loop function.

Circadian fluctuations in per mRNA and protein are central to the operation of a negative feedback loop that is necessary for setting the free-running period and for entraining the circadian oscillator to light-dark cycles. In this study, per mRNA cycling and locomotor activity rhythms were measured under different light and dark cycling regimes to determine how photoperiods affect the molecular feedback loop and circadian behavior, respectively. These experiments reveal that per mRNA peaks in abundance 4 h after lights-off in photoperiods of < or = 16 h, that, phase shifts in per mRNA cycling and behavioral rhythmicity occur rapidly after flies are transferred from one photoperiod to another, and that photoperiods longer than 20 h abolish locomotor activity rhythms and leave per mRNA at a median constitutive level. These results indicate that the per feedback loop uses lights-off as a phase reference point and suggest (along with previous findings for per01 and tim01) that per mRNA cycling is not regulated via simple negative feedback from the per protein.

Developmental state and the circadian clock interact to influence the timing of eclosion in Drosophila melanogaster.

In Drosophila melanogaster, the emergence of adults from their pupal cases (eclosion) is gated by the circadian clock such that it occurs during a window of approximately 8-10 h starting 1-2 h before lights-on in 12-h light:12-h dark cycles (LD). This gate is shifted several hours earlier by the clock mutant per(s), indicating that the clock controls the phase of eclosion under these conditions. Both the day and the time of eclosion are determined by the interplay between developmental state and the circadian clock. At a certain phase of the circadian cycle, the circadian clock, either directly or through some circadian clock-controlled mechanism, measures development state, and those pharate adults that have reached a certain developmental state by this phase eclose during the first available gate, while those that have not wait until a subsequent gate. Using wing pigmentation as a late developmental state marker, an early boundary for when the circadian clock assesses developmental state occurs roughly at the time when lights go out during LD cycles. This event is shifted several hours earlier in per(s), showing that it is under circadian control. A fly's developmental state at the time of developmental assessment also influences when eclosion will occur (during the gate) in that flies whose wings have become pigmented early (12-24 h before assessment) will eclose earlier in the gate than those whose wings become pigmented late (0-12 h before assessment). These data suggest that the circadian clock (or some clock-controlled mechanism) measures developmental state (wing pigmentation) in wild-type flies between lights-off and expression of the first clock-regulated marker approximately 4-5 h before eclosion and that the developmental state of the fly determines both which gate is chosen for eclosion and when eclosion occurs during that gate.

Temporal and spatial expression of an adult cuticle protein gene from Drosophila suggests that its protein product may impart some specialized cuticle function.

An adult cuticle protein gene (Dacp-1) from Drosophila melanogaster has been isolated and characterized. This gene was classified as an adult cuticle protein gene because it maintains the conserved structure of other cuticle protein genes, the sequence of its conceptual translation product contains a repeated motif that is found almost exclusively in a subset of adult cuticle proteins from Locust migratoria, and the gene is expressed in the epidermis underlying the head and thoracic cuticle. The bulk of Dacp-1 expression starts approximately 72 hr after pupariation, peaks approximately 12 hr after eclosion, and decreases thereafter to undetectable levels by 3 days after eclosion. The stage specificity and spatial restriction of Dacp-1 expression as well as the physical properties of its conceptual translation product suggest that it may be involved in some specialized function such as thickening of the adult cuticle.