François Rouyer

Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain

In Drosophila, a ‘clock’ situated in the brain controls circadian rhythms of locomotor activity. This clock relies on several groups of neurons that express the Period (PER) protein, including the ventral lateral neurons (LNvs), which express the Pigment-dispersing factor (PDF) neuropeptide, and the PDF-negative dorsal lateral neurons (LNds). In normal cycles of day and night, adult flies exhibit morning and evening peaks of activity; however, the contribution of the different clock neurons to the rest–activity pattern remains unknown. Here, we have used targeted expression of PER to restore the clock function of specific subsets of lateral neurons in arrhythmic per0 mutant flies. We show that PER expression restricted to the LNvs only restores the morning activity, whereas expression of PER in both the LNvs and LNds also restores the evening activity. This provides the first neuronal bases for ‘morning’ and ‘evening’ oscillators in the Drosophila brain. Furthermore, we show that the LNvs alone can generate 24 h activity rhythms in constant darkness, indicating that the morning oscillator is sufficient to drive the circadian system.

The F-box protein slimb controls the levels of clock proteins period and timeless.

The Drosophila circadian clock is driven by daily fluctuations of the proteins Period and Timeless, which associate in a complex and negatively regulate the transcription of their own genes. Protein phosphorylation has a central role in this feedback loop, by controlling Per stability in both cytoplasmic and nuclear compartments as well as Per/Tim nuclear transfer. However, the pathways regulating degradation of phosphorylated Per and Tim are unknown. Here we show that the product of the slimb (slmb) gene—a member of the F-box/WD40 protein family of the ubiquitin ligase SCF complex that targets phosphorylated proteins for degradation—is an essential component of the Drosophila circadian clock. slmb mutants are behaviourally arrhythmic, and can be rescued by targeted expression of Slmb in the clock neurons. In constant darkness, highly phosphorylated forms of the Per and Tim proteins are constitutively present in the mutants, indicating that the control of their cyclic degradation is impaired. Because levels of Per and Tim oscillate in slmb mutants maintained in light:dark conditions, light- and clock-controlled degradation of Per and Tim do not rely on the same mechanisms.

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.

Novel Features of Cryptochrome-Mediated Photoreception in the Brain Circadian Clock of Drosophila

In Drosophila, light affects circadian behavioral rhythms via at least two distinct mechanisms. One of them relies on the visual phototransduction cascade. The other involves a presumptive photopigment, cryptochrome (cry), expressed in lateral brain neurons that control behavioral rhythms. We show here that cry is expressed in most, if not all, larval and adult neuronal groups expressing the PERIOD (PER) protein, with the notable exception of larval dorsal neurons (DN2s) in which PER cycles in antiphase to all other known cells. Forcing cry expression in the larval DN2s gave them a normal phase of PER cycling, indicating that their unique antiphase rhythm is related to their lack of cry expression. We were able to directly monitor CRY protein in Drosophila brains in situ. It appeared highly unstable in the light, whereas in the dark, it accumulated in both the nucleus and the cytoplasm, including some neuritic projections. We also show that dorsal PER-expressing brain neurons, the adult DN1s, are the only brain neurons to coexpress the CRY protein and the photoreceptor differentiation factor GLASS. Studies of various visual system mutants and their combination with the cryb mutation indicated that the adult DN1s contribute significantly to the light sensitivity of the clock controlling activity rhythms, and that this contribution depends on CRY. Moreover, all CRY-independent light inputs into this central behavioral clock were found to require the visual system. Finally, we show that the photoreceptive DN1 neurons do not behave as autonomous oscillators, because their PER oscillations in constant darkness rapidly damp out in the absence of pigment-dispersing-factor signaling from the ventral lateral neurons.

Peptidergic clock neurons in Drosophila: Ion transport peptide and short neuropeptide F in subsets of dorsal and ventral lateral neurons

About 150 clock neurons are clustered in different groups in the brain of Drosophila. Among these clock neurons, some pigment‐dispersing factor (PDF)‐positive and PDF‐negative lateral neurons (LNs) are principal oscillators responsible for bouts of activity in the morning and evening, respectively. The full complement of neurotransmitters in these morning and evening oscillators is not known. By using a screen for candidate neuromediators in clock neurons, we discovered ion transport peptide (ITP) and short neuropeptide F (sNPF) as novel neuropeptides in subpopulations of dorsal (LNds) and ventral (s‐LNvs) LNs. Among the six LNds, ITP was found in one that coexpresses long neuropeptide F (NPF) and cryptochrome. We detected sNPF in two LNds that also express cryptochrome; these cells are distinct from three LNds expressing NPF. Thus, we have identified neuropeptides in five of the six LNds. The three LNds expressing cryptochrome, with either ITP or sNPF, are the only ones with additional projections to the accessory medulla. Among the five s‐LNvs in the adult brain, ITP was detected in the fifth neuron that is devoid of PDF and sNPF in the four neurons that also express PDF. By using a choline acetyltransferase (Cha) Gal4, we detected Cha expression in the two sNPF producing LNds and in the fifth s‐LNv. In the larval brain, two of the four PDF‐producing s‐LNvs coexpress sNPF. Our findings emphasize that the LNds are heterogeneous both anatomically and with respect to content of neuropeptides, cryptochrome, and other markers and suggest diverse functions of these neurons. J. Comp. Neurol. 516:59–73, 2009.

Effects of circadian mutations and LD periodicity on the life span of Drosophila melanogaster.

The pervasive occurrence of circadian clocks throughout the living world underlines their adaptive value. Nonetheless, there is surprisingly little evidence for a negative impact, on any animal species, of a constant discrepancy between the environmental and endogenous periods. Male Drosophila melanogaster per mutants with altered circadian periods were compared to the wild type in two different LD schedules. Life span was used as a global index of physiological adaptation. The life span of the mutants was significantly reduced by up to 15% for the flies whose period differs most from that of the wild type. A reduction was observed even when flies were kept in an LD schedule fitting a mutant period. The LD schedule made no significant difference on its own, but the authors found evidence for an interaction between genotype and LD schedule in determining life span. These results are consistent with the importance of the circadian clock in maintaining internal temporal order independent of environ-mental cycles. Nonetheless, a large difference between the environmental and endogenous periods has a measurable impact.

The Clockwork Orange Drosophila Protein Functions as Both an Activator and a Repressor of Clock Gene Expression

The Drosophila clock relies on transcriptional feedback loops that generate daily oscillations of the clock gene expression at mRNA and protein levels. In the evening, the CLOCK (CLK) and CYCLE (CYC) basic helix-loop-helix (bHLH) PAS-domain transcription factors activate the expression of the period (per) and timeless (tim) genes. Posttranslational modifications delay the accumulation of PER and TIM, which inhibit CLK/CYC activity in the late night. We show here that a null mutant of the clockwork orange (cwo) gene encoding a bHLH orange-domain putative transcription factor displays long-period activity rhythms. cwo loss of function increases cwo mRNA levels but reduces mRNA peak levels of the 4 described CLK/CYC targets, inducing an almost complete loss of their cycling. In addition, the absence of CWO induces alterations of PER and CLK phosphorylation cycles. Our results indicate that, in vivo, CWO modulates clock gene expression through both repressor and activator transcriptional functions.

Circadian rhythms of locomotor activity in Drosophila

Drosophila is by far the most advanced model to understand the complex biochemical interactions upon which circadian clocks rely. Most of the genes that have been characterized so far were isolated through genetic screens using the locomotor activity rhythms of the adults as a circadian output. In addition, new techniques are available to deregulate gene expression in specific cells, allowing to analyze the growing number of developmental genes that also play a role as clock genes. However, one of the major challenges in circadian biology remains to properly interpret complex behavioral data and use them to fuel molecular models. This review tries to describe the problems that clockwatchers have to face when using Drosophila activity rhythms to understand the multiple facets of circadian function.

A new gene encoding a putative transcription factor regulated by the Drosophila circadian clock

Circadian rhythms of locomotor activity and eclosion in Drosophila depend upon the reciprocal autoregulation of the period (per) and timeless (tim) genes. As part of this regulatory loop, per and tim mRNA levels oscillate in a circadian fashion. Other cycling transcripts may participate in this central pacemaker mechanism or represent outputs of the clock. In this paper, we report the isolation of Crg‐1, a new circadianly regulated gene. Like per and tim transcript levels, Crg‐1 transcript levels oscillate with a 24 h period in light:dark (LD) conditions, with a maximal abundance at the beginning of the night. These oscillations persist in complete darkness and depend upon per and tim proteins. The putative CRG‐1 proteins show some sequence similarity with the DNA‐binding domain of the HNF3/fork head family of transcription factors. In the adult head, in situ hybridization analysis reveals that per and Crg‐1 have similar expression patterns in the eyes and optic lobes.

PDF-modulated visual inputs and cryptochrome define diurnal behavior in Drosophila

Morning and evening circadian oscillators control the bimodal activity of Drosophila in light-dark cycles. The lateral neurons evening oscillator (LN-EO) is important for promoting diurnal activity at dusk. We found that the LN-EO autonomously synchronized to light-dark cycles through either the cryptochrome (CRY) that it expressed or the visual system. In conditions in which CRY was not activated, flies depleted for pigment-dispersing factor (PDF) or its receptor lost the evening activity and displayed reversed PER oscillations in the LN-EO. Rescue experiments indicated that normal PER cycling and the presence of evening activity relied on PDF secretion from the large ventral lateral neurons and PDF receptor function in the LN-EO. The LN-EO thus integrates light inputs and PDF signaling to control Drosophila diurnal behavior, revealing a new clock-independent function for PDF.