Timed receptor tyrosine kinase signaling couples the central and a peripheral circadian clock in Drosophila.

Circadian clocks impose daily periodicities to behavior, physiology, and metabolism. This control is mediated by a central clock and by peripheral clocks, which are synchronized to provide the organism with a unified time through mechanisms that are not fully understood. Here, we characterized in Drosophila the cellular and molecular mechanisms involved in coupling the central clock and the peripheral clock located in the prothoracic gland (PG), which together control the circadian rhythm of emergence of adult flies. The time signal from central clock neurons is transmitted via small neuropeptide F (sNPF) to neurons that produce the neuropeptide Prothoracicotropic Hormone (PTTH), which is then translated into daily oscillations of Ca2+ concentration and PTTH levels. PTTH signaling is required at the end of metamorphosis and transmits time information to the PG through changes in the expression of the PTTH receptor tyrosine kinase (RTK), TORSO, and of ERK phosphorylation, a key component of PTTH transduction. In addition to PTTH, we demonstrate that signaling mediated by other RTKs contributes to the rhythmicity of emergence. Interestingly, the ligand to one of these receptors (Pvf2) plays an autocrine role in the PG, which may explain why both central brain and PG clocks are required for the circadian gating of emergence. Our findings show that the coupling between the central and the PG clock is unexpectedly complex and involves several RTKs that act in concert and could serve as a paradigm to understand how circadian clocks are coordinated.

Functional Analyses of Four Cryptochromes From Aquatic Organisms After Heterologous Expression in Drosophila melanogaster Circadian Clock Cells.

Cryptochromes (Crys) represent a multi-facetted class of proteins closely associated with circadian clocks. They have been shown to function as photoreceptors but also to fulfill light-independent roles as transcriptional repressors within the negative feedback loop of the circadian clock. In addition, there is evidence for Crys being involved in light-dependent magneto-sensing, and regulation of neuronal activity in insects, adding to the functional diversity of this cryptic protein class. In mammals, Crys are essential components of the circadian clock, but their role in other vertebrates is less clear. In invertebrates, Crys can function as circadian photoreceptors, or as components of the circadian clock, while in some species, both light-receptive and clock factor roles coexist. In the current study, we investigate the function of Cry proteins in zebrafish (Danio rerio), a freshwater teleost expressing 6 cry genes. Zebrafish peripheral circadian clocks are intrinsically light-sensitive, suggesting the involvement of Cry in light-resetting. Echinoderms (Strongylocentrotus purpuratus) represent the only class of deuterostomes that possess an orthologue (SpuCry) of the light-sensitive Drosophila melanogaster Cry, which is an important component of the light-resetting pathway, but also works as transcriptional repressor in peripheral clocks of fruit flies. We therefore investigated the potential of different zebrafish cry genes and SpuCry to replace the light-resetting and repressor functions of Drosophila Cry by expressing them in fruit flies lacking endogenous cry function. Using various behavioral and molecular approaches, we show that most Cry proteins analyzed are able to fulfill circadian repressor functions in flies, except for one of the zebrafish Crys, encoded by cry4a. Cry4a also shows a tendency to support light-dependent Cry functions, indicating that it might act in the light-input pathway of zebrafish.

Sensitive Timing: A Reappraisal of Chronobiology's Foundational Texts.

The origin of experimental chronobiology can be traced to observations made in the 18th and 19th centuries on the sensitive plant Mimosa, which were described in two seminal reports: Jean-Jacques d'Ortous de Mairan's "Observation Botanique" (A Botanical Observation) and Augustin Pyramus de Candolle's "Du sommeil des feuilles" (On the sleep of leaves). Both report observations of the striking daily closing and opening of Mimosa leaves in controlled environments. This review presents translations of both texts with the aim of staying as faithful as possible to the original French texts. We also present the historical context in which these texts were written and link them to subsequent experiments that aimed at testing the veracity of their central conclusions. In particular, we definitely establish that Mairan himself presented his work to the French Royal Academy of Sciences, while the published report of his observation was authored by Fontenelle, the Secretary of the Academy. In addition, we offer a translation of Mairan's own presentation, based on the hand-written minutes of the academy. Finally, we discuss the decades of work on plant rhythms that laid the foundation for modern experimental chronobiology, including translations and discussion of the insightful and prescient reports by Charles François de Cisternay Dufay, Henri Louis Duhamel du Monceau, Johann Gottfried Zinn, and Wilhelm Pfeffer, which describe their efforts to reproduce and extend Mairan's pioneering observations.

A novel period mutation implicating nuclear export in temperature compensation of the Drosophila circadian clock.

Circadian clocks are self-sustained molecular oscillators controlling daily changes of behavioral activity and physiology. For functional reliability and precision, the frequency of these molecular oscillations must be stable at different environmental temperatures, known as "temperature compensation." Despite being an intrinsic property of all circadian clocks, this phenomenon is not well understood at the molecular level. Here, we use behavioral and molecular approaches to characterize a novel mutation in the period (per) clock gene of Drosophila melanogaster, which alters a predicted nuclear export signal (NES) of the PER protein and affects temperature compensation. We show that this new perI530A allele leads to progressively longer behavioral periods and clock oscillations with increasing temperature in both clock neurons and peripheral clock cells. While the mutant PERI530A protein shows normal circadian fluctuations and post-translational modifications at cool temperatures, increasing temperatures lead to both severe amplitude dampening and hypophosphorylation of PERI530A. We further show that PERI530A displays reduced repressor activity at warmer temperatures, presumably because it cannot inactivate the transcription factor CLOCK (CLK), indicated by temperature-dependent altered CLK post-translational modification in perI530A flies. With increasing temperatures, nuclear accumulation of PERI530A within clock neurons is increased, suggesting that wild-type PER is exported out of the nucleus at warm temperatures. Downregulating the nuclear export factor CRM1 also leads to temperature-dependent changes of behavioral rhythms, suggesting that the PER NES and the nuclear export of clock proteins play an important role in temperature compensation of the Drosophila circadian clock.

Light triggers a network switch between circadian morning and evening oscillators controlling behaviour during daily temperature cycles.

Proper timing of rhythmic locomotor behavior is the consequence of integrating environmental conditions and internal time dictated by the circadian clock. Rhythmic environmental input like daily light and temperature changes (called Zeitgeber) reset the molecular clock and entrain it to the environmental time zone the organism lives in. Furthermore, depending on the absolute temperature or light intensity, flies exhibit their main locomotor activity at different times of day, i.e., environmental input not only entrains the circadian clock but also determines the phase of a certain behavior. To understand how the brain clock can distinguish between (or integrate) an entraining Zeitgeber and environmental effects on activity phase, we attempted to entrain the clock with a Zeitgeber different from the environmental input used for phasing the behavior. 150 clock neurons in the Drosophila melanogaster brain control different aspects of the daily activity rhythms and are organized in various clusters. During regular 12 h light: 12 h dark cycles at constant mild temperature (LD 25°C, LD being the Zeitgeber), so called morning oscillator (MO) neurons control the increase of locomotor activity just before lights-on, while evening oscillator (EO) neurons regulate the activity increase at the end of the day, a few hours before lights-off. Here, using 12 h: 12 h 25°C:16°C temperature cycles as Zeitgeber, we attempted to look at the impact of light on phasing locomotor behavior. While in constant light and 25°C:16°C temperature cycles (LLTC), flies show an unimodal locomotor activity peak in the evening, during the same temperature cycle, but in the absence of light (DDTC), the phase of the activity peak is shifted to the morning. Here, we show that the EO is necessary for synchronized behavior in LLTC but not for entraining the molecular clock of the other clock neuronal groups, while the MO controls synchronized morning activity in DDTC. Interestingly, our data suggest that the influence of the EO on the synchronization increases depending on the length of the photoperiod (constant light vs 12 h of light). Hence, our results show that effects of different environmental cues on clock entrainment and activity phase can be separated, allowing to decipher their integration by the circadian clock.

Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster.

Circadian clocks are highly conserved transcriptional regulators that control ~24 hr oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila, which we term Locally Activatable BioLuminescence, or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcriptional properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks. We found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. We also demonstrate that distinct clocks exhibit differences in their loss of oscillatory amplitude or their change in period, depending on their anatomical location, mutation, or fly age. Our results demonstrate that LABL is an effective tool that allows rapid, affordable, and direct real-time monitoring of individual clocks in vivo.

The daily rhythms in our lives are driven by biological mechanisms called circadian clocks. These biological clocks are protein machines found in almost every cell and organ of the body, in nearly all living things, from fungi and plants to fruit flies and humans. These clocks control 24-hour cycles of gene activity and behaviour, and are kept in-time by so-called 'master clocks' in the brain. Ideally, scientists would be able to observe how circadian clocks work in different parts of the brain in a living animal and track changes throughout the day, as the animal performs different behaviours. However, the tools that are currently available to study circadian clocks do not allow this. To overcome this difficulty, Johnstone et al. used fruit flies to develop a new method that allows scientists to measure the oscillations of the circadian clocks in the brain in real time. Circadian clocks are composed of proteins called 'transcription factors' that activate different genes throughout the day, producing different proteins at different times. Transcription factors control the activity of genes by binding to DNA sequences called 'promoters' and switching the genes regulated by these promoters on or off. Knowing this, Johnstone et al. engineered fruit flies to carry the gene that codes for a protein called luciferase, which emits light, and placed it under the control of the promoter for the period gene, a gene that is regulated by the circadian clock. To prevent all of the cells in the fly from producing luciferase any time the period promoter was active, Johnstone et al. placed a second gene between the promoter and the luciferase gene. This second gene contains 'stop' sequences that prevent luciferase from being produced as long as the second gene is present. Importantly, this gene can be genetically removed from specific cells in live flies, so only these cells will produce luciferase. When Johnstone et al. removed the second gene from specific cells in the fly brain that are involved in controlling behaviours related to the circadian clocks, these cells started emitting light in cycles that reproduced the activity of the circadian clocks. Thus, by monitoring how the brightness of luciferase changed throughout the day in these flies, Johnstone et al. were able to reveal how the circadian clocks work in different parts of the fly brain. They found that each clock had slightly different cycling lengths, suggesting that the clocks work differently in different parts of the brain to control behaviour. Interestingly, Johnstone et al. found that if a key gene responsible for communication between cells was mutated, the effects of the mutation also varied in different parts of the brain. This suggests that different clocks respond differently to communication cues. Additionally, the results showed that circadian clock activity also changed with age: older flies had weaker circadian behaviours ­ fewer changes in both behavioural and genetic activity levels between the day and night ­ than younger animals. Johnstone et al.'s approach makes it possible to track a living animal's circadian clocks in different parts of the brain and in different organs in real time without the need to dissect the animal. In the future, this method will help scientists understand the links between different circadian clocks, the genes associated with them, and the behaviours they control.

A natural timeless polymorphism allowing circadian clock synchronization in "white nights".

Daily temporal organisation offers a fitness advantage and is determined by an interplay between environmental rhythms and circadian clocks. While light:dark cycles robustly synchronise circadian clocks, it is not clear how animals experiencing only weak environmental cues deal with this problem. Like humans, Drosophila originate in sub-Saharan Africa and spread North up to the polar circle, experiencing long summer days or even constant light (LL). LL disrupts clock function, due to constant activation of CRYPTOCHROME, which induces degradation of the clock protein TIMELESS (TIM), but temperature cycles are able to overcome these deleterious effects of LL. We show here that for this to occur a recently evolved natural timeless allele (ls-tim) is required, encoding the less light-sensitive L-TIM in addition to S-TIM, the only form encoded by the ancient s-tim allele. We show that only ls-tim flies can synchronise their behaviour to semi-natural conditions typical for Northern European summers, suggesting that this functional gain is driving the Northward ls-tim spread.

The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days.

Cation chloride cotransporters (CCCs) regulate intracellular chloride ion concentration ([Cl-]i) within neurons, which can reverse the direction of the neuronal response to the neurotransmitter GABA.1 Na+ K+ Cl- (NKCC) and K+ Cl- (KCC) cotransporters transport Cl- into or out of the cell, respectively. When NKCC activity dominates, the resulting high [Cl-]i can lead to an excitatory and depolarizing response of the neuron upon GABAA receptor opening, while KCC dominance has the opposite effect.1 This inhibitory-to-excitatory GABA switch has been linked to seasonal adaption of circadian clock function to changing day length,2-4 and its dysregulation is associated with neurodevelopmental disorders such as epilepsy.5-8 In Drosophila melanogaster, constant light normally disrupts circadian clock function and leads to arrhythmic behavior.9 Here, we demonstrate a function for CCCs in regulating Drosophila locomotor activity and GABA responses in circadian clock neurons because alteration of CCC expression in circadian clock neurons elicits rhythmic behavior in constant light. We observed the same effects after downregulation of the Wnk and Fray kinases, which modulate CCC activity in a [Cl-]i-dependent manner. Patch-clamp recordings from the large LNv clock neurons show that downregulation of KCC results in a more positive GABA reversal potential, while KCC overexpression has the opposite effect. Finally, KCC and NKCC downregulation reduces or increases morning behavioral activity during long photoperiods, respectively. In summary, our results support a model in which the regulation of [Cl-]i by a KCC/NKCC/Wnk/Fray feedback loop determines the response of clock neurons to GABA, which is important for adjusting behavioral activity to constant light and long-day conditions.

Evolution of casein kinase 1 and functional analysis of new doubletime mutants in Drosophila.

Circadian clocks are timing devices that rhythmically adjust organism's behavior, physiology, and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks rely on several interlocked transcription-translation feedback loops, where protein stability is the key part of the delay between transcription and the appearance of the mature proteins within the feedback loops. In bilaterian animals, including mammals and insects, the circadian clock depends on a homologous set of proteins. Despite mostly conserved clock components among the fruit fly Drosophila and mammals, several lineage-specific differences exist. Here we have systematically explored the evolution and sequence variability of insect DBT proteins and their vertebrate homologs casein kinase 1 delta (CKIδ) and epsilon (CKIε), dated the origin and separation of CKIδ from CKIε, and identified at least three additional independent duplications of the CKIδ/ε gene in Petromyzon, Danio, and Xenopus. We determined conserved regions in DBT specific to Diptera, and functionally tested a subset of those in D. melanogaster. Replacement of Lysine K224 with acidic residues strongly impacts the free-running period even in heterozygous flies, whereas homozygous mutants are not viable. K224D mutants have a temperature compensation defect with longer free-running periods at higher temperatures, which is exactly the opposite trend of what was reported for corresponding mammalian mutants. All DBTs of dipteran insects contain the NKRQK motif at positions 220-224. The occurrence of this motif perfectly correlates with the presence of BRIDE OF DOUBLETIME, BDBT, in Diptera. BDBT is a non-canonical FK506-binding protein that physically interacts with Drosophila DBT. The phylogeny of FK506-binding proteins suggests that BDBT is either absent or highly modified in non-dipteran insects. In addition to in silico analysis of DBT/CKIδ/ε evolution and diversity, we have identified four novel casein kinase 1 genes specific to the Drosophila genus.

Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity.

In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, we performed a systematic RNAi screen in human cells and identified protein phosphatase 4 (PPP4) with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila Genetic depletion of PPP4 shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the "kamikaze" properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1δ-mediated inhibition of CLOCK/BMAL1.

Peripheral Sensory Organs Contribute to Temperature Synchronization of the Circadian Clock in Drosophila melanogaster.

Circadian clocks are cell-autonomous endogenous oscillators, generated and maintained by self-sustained 24-h rhythms of clock gene expression. In the fruit fly Drosophila melanogaster, these daily rhythms of gene expression regulate the activity of approximately 150 clock neurons in the fly brain, which are responsible for driving the daily rest/activity cycles of these insects. Despite their endogenous character, circadian clocks communicate with the environment in order to synchronize their self-sustained molecular oscillations and neuronal activity rhythms (internal time) with the daily changes of light and temperature dictated by the Earth's rotation around its axis (external time). Light and temperature changes are reliable time cues (Zeitgeber) used by many organisms to synchronize their circadian clock to the external time. In Drosophila, both light and temperature fluctuations robustly synchronize the circadian clock in the absence of the other Zeitgeber. The complex mechanisms for synchronization to the daily light-dark cycles are understood with impressive detail. In contrast, our knowledge about how the daily temperature fluctuations synchronize the fly clock is rather limited. Whereas light synchronization relies on peripheral and clock-cell autonomous photoreceptors, temperature input to the clock appears to rely mainly on sensory cells located in the peripheral nervous system of the fly. Recent studies suggest that sensory structures located in body and head appendages are able to detect temperature fluctuations and to signal this information to the brain clock. This review will summarize these studies and their implications about the mechanisms underlying temperature synchronization.

The Effect of General Anaesthesia on Circadian Rhythms in Behaviour and Clock Gene Expression of Drosophila melanogaster.

General anaesthesia (GA) is implicated as a cause of postoperative sleep disruption and fatigue with part of the disturbance being attributed to a shift of the circadian clock. In this study, Drosophila melanogaster was used as a model to determine how Isoflurane affects the circadian clock at the behavioural and molecular levels. We measured the response of the clock at both of these levels caused by different durations and different concentrations of Isoflurane at circadian time 4 (CT4). Once characterized, we held the duration and concentration constants (at 2% in air for 6 h) and calculated the phase responses over the entire circadian cycle in both activity and period expression. Phase advances in behaviour were observed during the subjective day, whereas phase delays were associated with subjective night time GA interventions. The corresponding pattern of gene expression preceded the behavioural pattern by approximately four hours. We discuss the implications of this effect for clinical and research practice.

A Robust and Self-Sustained Peripheral Circadian Oscillator Reveals Differences in Temperature Compensation Properties with Central Brain Clocks.

Circadian clocks are characterized by three properties: they run in constant conditions with a period of ∼24 h, synchronize to the environmental cycles of light and temperature, and are temperature compensated, meaning they do not speed up with temperature. Central brain clocks regulate daily activity rhythms, whereas peripheral clocks are dispersed throughout the body of insects and vertebrates. Using a set of luciferase reporter genes, we show that Drosophila peripheral clocks are self-sustained but over-compensated, i.e., they slow down with increasing temperature. In contrast, central clock neurons in the fly brain, both in intact flies and in cultured brains, show accurate temperature compensation. Although this suggests that neural network properties contribute to temperature compensation, the circadian neuropeptide Pigment Dispersing Factor (PDF) is not required for temperature-compensated oscillations in brain clock neurons. Our findings reveal a fundamental difference between central and peripheral clocks, which likely also applies for vertebrate clocks.

Publisher Correction: Methylation deficiency disrupts biological rhythms from bacteria to humans.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Light Sampling via Throttled Visual Phototransduction Robustly Synchronizes the Drosophila Circadian Clock.

The daily changes of light and dark exemplify a prominent cue for the synchronization of circadian clocks with the environment. The match between external and internal time is crucial for the fitness of organisms, and desynchronization has been linked to numerous physical and mental health problems. Organisms therefore developed complex and not fully understood mechanisms to synchronize their circadian clock to light. In mammals and in Drosophila, both the visual system and non-image-forming photoreceptors contribute to circadian clock resetting. In Drosophila, light-dependent degradation of the clock protein TIMELESS by the blue light photoreceptor Cryptochrome is considered the main mechanism for clock synchronization, although the visual system also contributes. To better understand the visual system contribution, we generated a genetic variant exhibiting extremely slow phototransduction kinetics, yet normal sensitivity. In this variant, the visual system is able to contribute its full share to circadian clock entrainment, both with regard to behavioral and molecular light synchronization. This function depends on an alternative phospholipase C-ß enzyme, encoded by PLC21C, presumably playing a dedicated role in clock resetting. We show that this pathway requires the ubiquitin ligase CULLIN-3, possibly mediating CRY-independent degradation of TIMELESS during light:dark cycles. Our results suggest that the PLC21C-mediated contribution to circadian clock entrainment operates on a drastically slower timescale compared with fast, norpA-dependent visual phototransduction. Our findings are therefore consistent with the general idea that the visual system samples light over prolonged periods of time (h) in order to reliably synchronize their internal clocks with the external time.

Methylation deficiency disrupts biological rhythms from bacteria to humans.

The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies.

New Drosophila Circadian Clock Mutants Affecting Temperature Compensation Induced by Targeted Mutagenesis of Timeless.

Drosophila melanogaster has served as an excellent genetic model to decipher the molecular basis of the circadian clock. Two key proteins, PERIOD (PER) and TIMELESS (TIM), are particularly well explored and a number of various arrhythmic, slow, and fast clock mutants have been identified in classical genetic screens. Interestingly, the free running period (tau, τ) is influenced by temperature in some of these mutants, whereas τ is temperature-independent in other mutant lines as in wild-type flies. This, so-called "temperature compensation" ability is compromised in the mutant timeless allele "ritsu" (tim rit ), and, as we show here, also in the tim blind allele, mapping to the same region of TIM. To test if this region of TIM is indeed important for temperature compensation, we generated a collection of new mutants and mapped functional protein domains involved in the regulation of τ and in general clock function. We developed a protocol for targeted mutagenesis of specific gene regions utilizing the CRISPR/Cas9 technology, followed by behavioral screening. In this pilot study, we identified 20 new timeless mutant alleles with various impairments of temperature compensation. Molecular characterization revealed that the mutations included short in-frame insertions, deletions, or substitutions of a few amino acids resulting from the non-homologous end joining repair process. Our protocol is a fast and cost-efficient systematic approach for functional analysis of protein-coding genes and promoter analysis in vivo. Interestingly, several mutations with a strong temperature compensation defect map to one specific region of TIM. Although the exact mechanism of how these mutations affect TIM function is as yet unknown, our in silico analysis suggests they affect a putative nuclear export signal (NES) and phosphorylation sites of TIM. Immunostaining for PER was performed on two TIM mutants that display longer τ at 25°C and complete arrhythmicity at 28°C. Consistently with the behavioral phenotype, PER immunoreactivity was reduced in circadian clock neurons of flies exposed to elevated temperatures.

Temperature synchronization of the Drosophila circadian clock protein PERIOD is controlled by the TRPA channel PYREXIA.

Circadian clocks are endogenous molecular oscillators that temporally organize behavioral activity thereby contributing to the fitness of organisms. To synchronize the fly circadian clock with the daily fluctuations of light and temperature, these environmental cues are sensed both via brain clock neurons, and by light and temperature sensors located in the peripheral nervous system. Here we demonstrate that the TRPA channel PYREXIA (PYX) is required for temperature synchronization of the key circadian clock protein PERIOD. We observe a molecular synchronization defect explaining the previously reported defects of pyx mutants in behavioral temperature synchronization. Surprisingly, surgical ablation of pyx-mutant antennae partially rescues behavioral synchronization, indicating that antennal temperature signals are modulated by PYX function to synchronize clock neurons in the brain. Our results suggest that PYX protects antennal neurons from faulty signaling that would otherwise interfere with temperature synchronization of the circadian clock neurons in the brain.

Development of the Molecular Circadian Clock and Its Light Sensitivity in Drosophila Melanogaster.

The importance of the circadian clock for the control of behavior and physiology is well established but how and when it develops is not fully understood. Here the initial expression pattern of the key clock gene period was recorded in Drosophila from embryos in vivo, using transgenic luciferase reporters. PERIOD expression in the presumptive central-clock dorsal neurons started to oscillate in the embryo in constant darkness. In behavioral experiments, a single 12-h light pulse given during the embryonic stage synchronized adult activity rhythms, implying the early development of entrainment mechanisms. These findings suggest that the central clock is functional already during embryogenesis. In contrast to central brain expression, PERIOD in the peripheral cells or their precursors increased during the embryonic stage and peaked during the pupal stage without showing circadian oscillations. Its rhythmic expression only initiated in the adult. We conclude that cyclic expression of PERIOD in the central-clock neurons starts in the embryo, presumably in the dorsal neurons or their precursors. It is not until shortly after eclosion when cyclic and synchronized expression of PERIOD in peripheral tissues commences throughout the animal.

DN1p or the "Fluffy" Cerberus of Clock Outputs.

Drosophila melanogaster is a powerful genetic model to study the circadian clock. Recently, three drosophilists received the Nobel Prize for their intensive past and current work on the molecular clockwork (Nobel Prize 2017). The Drosophila brain clock is composed of about 150 clock neurons distributed along the lateral and dorsal regions of the protocerebrum. These clock neurons control the timing of locomotor behaviors. In standard light-dark (LD) conditions (12-12 h and constant 25°C), flies present a bi-modal locomotor activity pattern controlled by the clock. Flies increase their movement just before the light-transitions, and these behaviors are therefore defined as anticipatory. Two neuronal oscillators control the morning and evening anticipation. Knowing that the molecular clock cycles in phase in all clock neurons in the brain in LD, how can we explain the presence of two behavioral activity peaks separated by 12 h? According to one model, the molecular clock cycles in phase in all clock neurons, but the neuronal activity cycles with a distinct phase in the morning and evening oscillators. An alternative model takes the environmental condition into consideration. One group of clock neurons, the dorso-posterior clock neurons DN1p, drive two peaks of locomotor activity in LD even though their neuronal activity cycles with the same phase (late night/early morning). Interestingly, the locomotor outputs they control differ in their sensitivity to light and temperature. Hence, they must drive outputs to different neuropil regions in the brain, which also receive different inputs. Since 2010 and the presentation of the first specific DN1p manipulations, many studies have been performed to understand the role of this group of neurons in controlling locomotor behaviors. Hence, we review what we know about this heterogeneous group of clock neurons and discuss the second model to explain how clock neurons that oscillate with the same phase can drive behaviors at different times of the day.