Circadian Rhythms In Drosophila Research Articles

Neural Stimulation during Drosophila Activity Monitor (DAM)-Based Studies of Sleep and Circadian Rhythms in Drosophila melanogaster.

Sleep is a fundamental feature of life for virtually all multicellular animals, but many questions remain about how sleep is regulated by circadian rhythms, homeostatic sleep drive that builds up with wakefulness, and modifying factors such as hunger or social interactions, as well as about the biological functions of sleep. Substantial headway has been made in the study of both circadian rhythms and sleep in the fruit fly Drosophila melanogaster, much of it through studies of individual fly activity using Drosophila activity monitors (DAMs). Here, we describe approaches for the activation of specific neurons of interest using optogenetics (involving genetic modifications that allow for light-based neuronal activation) and thermogenetics (involving genetic modifications that allow for temperature-based neuronal activation) so that researchers can evaluate the roles of those neurons in controlling rest and activity behavior. In this protocol, we describe how to set up a rig for simultaneous optogenetic or thermogenetic stimulation and activity monitoring for analysis of sleep and circadian rhythms in Drosophila, how to raise appropriate flies, and how to perform the experiment. This protocol will allow researchers to assess the causative role in the regulation of sleep and activity rhythms of any genetically tractable subset of cells.

MiR-277 regulates the phase of circadian activity-rest rhythm in Drosophila melanogaster.

Circadian clocks temporally organize behaviour and physiology of organisms with a rhythmicity of about 24h. In Drosophila, the circadian clock is composed of mainly four clock genes: period (per), timeless (tim), Clock (Clk) and cycle (cyc) which constitutes the transcription-translation feedback loop. The circadian clock is further regulated via post-transcriptional and post-translational mechanisms among which microRNAs (miRNAs) are well known post-transcriptional regulatory molecules. Here, we identified and characterized the role of miRNA-277 (miR-277) expressed in the clock neurons in regulating the circadian rhythm. Downregulation of miR-277 in the pacemaker neurons expressing circadian neuropeptide, pigment dispersing factor (PDF) advanced the phase of the morning activity peak under 12h light: 12h dark cycles (LD) at lower light intensities and these flies exhibited less robust rhythms compared to the controls under constant darkness. In addition, downregulation of miR-277 in the PDF expressing neurons abolished the Clk gene transcript oscillation under LD. Our study points to the potential role of miR-277 in fine tuning the Clk expression and in maintaining the phase of the circadian rhythm in Drosophila.

TIMELESS promotes the proliferation and migration of lung adenocarcinoma cells by activating EGFR through AMPK and SPHK1 regulation

BackgroundLung adenocarcinoma (LUAD) has high morbidity and is prone to recurrence. TIMELESS (TIM), which regulates circadian rhythms in Drosophila, is highly expressed in various tumors. Its role in LUAD has gained attention, but the detailed function and mechanism have not been clarified completely at present. MethodsTumor samples from patients with LUAD patient data from public databases were used to confirm the relationship of TIM expression with lung cancer. LUAD cell lines were used and siRNA of TIM was adopted to knock down TIM expression in LUAD cells, and further cell proliferation, migration and colony formation were analyzed. By using Western blot and qPCR, we detected the influence of TIM on epidermal growth factor receptor (EGFR), sphingosine kinase 1 (SPHK1) and AMP-activated protein kinase (AMPK). With proteomics analysis, we comprehensively inspected the different changed proteins influenced by TIM and did global bioinformatic analysis. ResultsWe found that TIM expression was elevated in LUAD and that this high expression was positively correlated with more advanced tumor pathological stages and shorter overall and disease-free survival. TIM knockdown inhibited EGFR activation and also AKT/mTOR phosphorylation. We also clarified that TIM regulated the activation of SPHK1 in LUAD cells. And with SPHK1 siRNA to knock down the expression level of SPHK1, we found that EGFR activation were inhibited greatly too. Quantitative proteomics techniques combined with bioinformatics analysis clarified the global molecular mechanisms regulated by TIM in LUAD. The results of proteomics suggested that mitochondrial translation elongation and termination were altered, which were closely related to the process of mitochondrial oxidative phosphorylation. We further confirmed that TIM knockdown reduced ATP content and promoted AMPK activation in LUAD cells. ConclusionsOur study revealed that siTIM could inhibit EGFR activation through activating AMPK and inhibiting SPHK1 expression, as well as influencing mitochondrial function and altering the ATP level; TIM's high expression in LUAD is an important factor and a potential key target in LUAD.

In vivo characterization of the maturation steps of PDF neuropeptide precursor in the Drosophila circadian pacemaker neurons.

Pigment Dispersing Factor (PDF), is a key signaling molecule coordinating the neuronal network associated with the circadian rhythms in Drosophila. The precursor (proPDF) of the mature PDF (mPDF) consists of two motifs, a larger PDF-associated peptide (PAP) and PDF. Through cleavage and amidation, the proPDF is predicted to produce cleaved-PAP (cPAP) and mPDF. To delve into the in vivo mechanisms underlying proPDF maturation, we generated various mutations that eliminate putative processing sites and then analyzed the effect of each mutation on the production of cPAP and mPDF by four different antibodies in both ectopic and endogenous conditions. We also assessed the knockdown effects of processing enzymes on the proPDF maturation. At the functional level, circadian phenotypes were measured for all mutants and knockdown lines. As results, we confirm the roles of key enzymes and their target residues: Amontillado (Amon) for the cleavage at the consensus dibasic KR site, Silver (Svr) for the removal of C-terminal basic residues from the intermediates, PAP-KR and PDF-GK, derived from proPDF, and PHM (Peptidylglycine-α-hydroxylating monooxygenase) for the amidation of PDF. Our results suggest that the C-terminal amidation occurs independently of proPDF cleavage. Moreover, PAP domain is important for the proPDF trafficking into the secretory vesicles and a close association between cPAP and mPDF following cleavage seems required for their stability within the vesicles. These studies highlight the biological significance of individual processing steps and the roles of the PAP for the stability and function of mPDF which is essential for the circadian clockworks.

Dibutyl phthalate disrupts conserved circadian rhythm in Drosophila and human cells

People are constantly exposed to phthalates, due to their common use in the production of plastics, pharmaceuticals, cosmetics and skin care products. The ability of phthalates to disrupt endocrine signaling, leading to developmental, reproductive and metabolic defects, has been studied, yet how phthalates interfere with these biological functions is still unclear. To uncover DBP interacting molecular pathways, we raised Drosophila melanogaster on food containing dibutyl phthalate (DBP) at various concentrations. Whole transcriptome analysis of adult Drosophila reveals that DBP exposure throughout development disrupts the expression of genes central to circadian rhythm regulation, including increased expression of vrille (vri, human NFIL3), timeless (tim, human TIMELESS) and period (per, human PER3), with decreased expression of Pigment-dispersing factor (Pdf). DBP exposure also alters the expression of the evolutionarily conserved nuclear receptor Hormone receptor-like in 38 (Hr38, human NR4A2), which is known to regulate Pdf expression. Furthermore, behavioral assays determined that exposing Drosophila to DBP throughout development modifies the circadian rhythm of adults. Although DBP inhibits the expression of signaling systems regulating vision, including Rh5 and Rh6, two light-sensing G-protein coupled receptors involved in the daily resetting of circadian rhythm, it does not influence eye development. Circadian rhythm genes are well conserved from flies to humans; therefore, we tested the effect of DBP exposure on human breast cells (MCF10A) and demonstrate that, similar to the fruit fly model, this exposure disrupts circadian rhythm (BMAL1 expression) at doses that promote the proliferation and migration ability of MCF10A cells. Our results are the first to provide comprehensive evidence that DBP interferes with circadian rhythm in both adult Drosophila and human cells, which may help to explain the broad physiological action of phthalates.

Dynamics of the Tyson–Hong–Thron–Novak circadian oscillator model

We study the dynamics of a circadian oscillator model proposed by Tyson, Hong, Thron and Novak. This model describes a molecular mechanism for the circadian rhythm in Drosophila. After giving a detailed study of its equilibria, we investigate the dynamics in the cases that the rate of mRNA degradation is sufficiently high or low. When the rate is sufficiently high, we prove that there are no periodic orbits in the region with biological meaning. When the rate is sufficiently low, this model is transformed into a slow–fast system. Then based on the geometric singular perturbation theory, we prove the existence of relaxation oscillations, canard explosion, saddle–node bifurcations, and the coexistence of two limit cycles in this model. These results are helpful to understand the effects of biophysical parameters on circadian oscillations. Finally, we give the biological interpretation of the results and point out that this model can be transformed into a Liénard-like equation, which could be helpful to investigate the dynamics of the general case.

Effective reduction of a three-dimensional circadian oscillator model

We investigate the dynamics of a three-dimensional system modeling a molecular mechanism for the circadian rhythm in Drosophila. We first prove the existence of a compact attractor in the region with biological meaning. Under the assumption that the dimerization reactions are fast, in this attractor we reduce the three-dimensional system to a simpler two-dimensional system on the persistent normally hyperbolic slow manifold.

Crystal structure and interactions of the Tof1-Csm3 (Timeless-Tipin) fork protection complex.

The Tof1–Csm3 fork protection complex has a central role in the replisome—it promotes the progression of DNA replication forks and protects them when they stall, while also enabling cohesion establishment and checkpoint responses. Here, I present the crystal structure of the Tof1–Csm3 complex from Chaetomium thermophilum at 3.1 Å resolution. The structure reveals that both proteins together form an extended alpha helical repeat structure, which suggests a mechanical or scaffolding role for the complex. Expanding on this idea, I characterize a DNA interacting region and a cancer-associated Mrc1 binding site. This study provides the molecular basis for understanding the functions of the Tof1–Csm3 complex, its human orthologue the Timeless–Tipin complex and additionally the Drosophila circadian rhythm protein Timeless.

Regulation of circadian locomotor rhythm by miR-263a

ABSTRACT The circadian clock is a rhythmic oscillator driving various physiological and behavioral processes that adapt to daily environmental changes. MicroRNAs (miRNAs) are a class of small non-coding RNAs that play an important role in post-transcriptional regulation. Although recent work has demonstrated that miRNAs are involved in development and disease, their function in the adult circadian locomotor rhythm still needs to be extensively characterized. In this study, we identified the miR-263a as an important regulator of Drosophila circadian locomotor rhythm, with miR-263a expression in LNv clock neurons. The overexpression of miR-263a in clock neurons lengthens the circadian period, while its mutant Bereft24 shortens the circadian period. In addition, downregulation of miR-263a also dramatically attenuates the morning activity peak as well as circadian rhythm robustness. Moreover, slo and homer are identified as the potential targets of the miR-263a. Together, these results establish that the miR-263a is an important regulator of circadian locomotor rhythm.

SUR-8 interacts with PP1-87B to stabilize PERIOD and regulate circadian rhythms in Drosophila.

Circadian rhythms are generated by endogenous pacemakers that rely on transcriptional-translational feedback mechanisms conserved among species. In Drosophila, the stability of a key pacemaker protein PERIOD (PER) is tightly controlled by changes in phosphorylation status. A number of molecular players have been implicated in PER destabilization by promoting PER progressive phosphorylation. On the other hand, there have been few reports describing mechanisms that stabilize PER by delaying PER hyperphosphorylation. Here we report that the protein Suppressor of Ras (SUR-8) regulates circadian locomotor rhythms by stabilizing PER. Depletion of SUR-8 from circadian neurons lengthened the circadian period by about 2 hours and decreased PER abundance, whereas its overexpression led to arrhythmia and an increase in PER. Specifically SUR-8 promotes the stability of PER through phosphorylation regulation. Interestingly, downregulation of the protein phosphatase 1 catalytic subunit PP1-87B recapitulated the phenotypes of SUR-8 depletion. We found that SUR-8 facilitates interactions between PP1-87B and PER. Depletion of SUR-8 decreased the interaction of PER and PP1-87B, which supports the role of SUR-8 as a scaffold protein. Interestingly, the interaction between SUR-8 and PER is temporally regulated: SUR-8 has more binding to PER at night than morning. Thus, our results indicate that SUR-8 interacts with PP1-87B to control PER stability to regulate circadian rhythms.

Drosophila PSI controls circadian period and the phase of circadian behavior under temperature cycle via tim splicing.

The Drosophila circadian pacemaker consists of transcriptional feedback loops subjected to post-transcriptional and post-translational regulation. While post-translational regulatory mechanisms have been studied in detail, much less is known about circadian post-transcriptional control. Thus, we targeted 364 RNA binding and RNA associated proteins with RNA interference. Among the 43 hits we identified was the alternative splicing regulator P-element somatic inhibitor (PSI). PSI regulates the thermosensitive alternative splicing of timeless (tim), promoting splicing events favored at warm temperature over those increased at cold temperature. Psi downregulation shortens the period of circadian rhythms and advances the phase of circadian behavior under temperature cycle. Interestingly, both phenotypes were suppressed in flies that could produce TIM proteins only from a transgene that cannot form the thermosensitive splicing isoforms. Therefore, we conclude that PSI regulates the period of Drosophila circadian rhythms and circadian behavior phase during temperature cycling through its modulation of the tim splicing pattern.

Expression of mutant CHMP2B linked to neurodegeneration in humans disrupts circadian rhythms in Drosophila.

Mutations in CHMP2B, an ESCRT‐III (endosomal sorting complexes required for transport) component, are associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Neurodegenerative disorders including FTD are also associated with a disruption in circadian rhythms, but the mechanism underlying this defect is not well understood. Here, we ectopically expressed the human CHMP2B variant associated with FTD (CHMP2BIntron5) in flies using the GMR‐GAL4 driver (GMR>CHMP2BIntron5) and analyzed their circadian rhythms at behavioral, cellular, and biochemical level. In GMR>CHMP2BIntron5 flies, we observed disrupted eclosion rhythms, shortened free‐running circadian locomotor period, and reduced levels of timeless (tim) mRNA—a circadian pacemaker gene. We also observed that the GMR‐GAL4 driver, primarily known for its expression in the retina, drives expression in a subset of tim expressing neurons in the optic lobe of the brain. The patterning of these GMR‐ and tim‐positive neurons in the optic lobe, which appears distinct from the putative clusters of circadian pacemaker neurons in the fly brain, was disrupted in GMR>CHMP2BIntron5 flies. These results demonstrate that CHMP2BIntron5 can disrupt the normal function of the circadian clock in Drosophila.

The Lysine Demethylase dKDM2 Is Non-essential for Viability, but Regulates Circadian Rhythms in Drosophila.

Post-translational modification of histones, such as histone methylation controlled by specific methyltransferases and demethylases, play critical roles in modulating chromatin dynamics and transcription in eukaryotes. Misregulation of histone methylation can lead to aberrant gene expression, thereby contributing to abnormal development and diseases such as cancer. As such, the mammalian lysine-specific demethylase 2 (KDM2) homologs, KDM2A and KDM2B, are either oncogenic or tumor suppressive depending on specific pathological contexts. However, the role of KDM2 proteins during development remains poorly understood. Unlike vertebrates, Drosophila has only one KDM2 homolog (dKDM2), but its functions in vivo remain elusive due to the complexities of the existing mutant alleles. To address this problem, we have generated two dKdm2 null alleles using the CRISPR/Cas9 technique. These dKdm2 homozygous mutants are fully viable and fertile, with no developmental defects observed under laboratory conditions. However, the dKdm2 null mutant adults display defects in circadian rhythms. Most of the dKdm2 mutants become arrhythmic under constant darkness, while the circadian period of the rhythmic mutant flies is approximately 1 h shorter than the control. Interestingly, lengthened circadian periods are observed when dKDM2 is overexpressed in circadian pacemaker neurons. Taken together, these results demonstrate that dKdm2 is not essential for viability; instead, dKDM2 protein plays important roles in regulating circadian rhythms in Drosophila. Further analyses of the molecular mechanisms of dKDM2 and its orthologs in vertebrates regarding the regulation of circadian rhythms will advance our understanding of the epigenetic regulations of circadian clocks.

TIMELESS confers cisplatin resistance in nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway and promoting the epithelial mesenchymal transition

This study investigated the expression, clinicopathological significance and mechanism of action of TIMELESS, a mammalian homolog of a Drosophila circadian rhythm gene, in nasopharyngeal carcinoma. Quantitative real-time PCR, western blotting and immunohistochemistry revealed TIMELESS was upregulated in NPC cell lines (n = 8 vs. NP69 cells), and freshly-frozen (n = 6) and paraffin-embedded human NPC specimens (n = 108 vs. normal samples/non-tumor cells). TIMELESS expression was associated with T category (P = 0.002), N category (P = 0.001), clinical stage (P < 0.001), metastasis (P = 0.047), vital status (P = 0.013) and serum Epstein–Barr DNA (P = 0.005). High TIMELESS expression was associated with poorer overall survival (80.7% vs. 95.9%; P = 0.004) and progression free survival (68.1% vs. 88.0%; P = 0.005). Univariate and multivariate analysis revealed TIMELESS was an independent prognostic factor for overall survival and progression free survival. Stable ectopic overexpression of TIMELESS in NPC cell lines conferred resistance to cisplatin-induced apoptosis in vitro and in vivo, promoted an epithelial-to-mesenchymal transition phenotype, and activated the Wnt/β-catenin pathway and downstream gene transcription; knockdown of TIMELESS had the opposite effects. TIMELESS may play a role in the development of NPC and could represent a valuable prognostic factor and potential therapeutic target.

MicroRNA-92a is a circadian modulator of neuronal excitability in Drosophila

Many biological and behavioural processes of animals are governed by an endogenous circadian clock, which is dependent on transcriptional regulation. Here we address post-transcriptional regulation and the role of miRNAs in Drosophila circadian rhythms. At least six miRNAs show cycling expression levels within the pigment dispersing factor (PDF) cell-pacemaker neurons; only mir-92a peaks during the night. In vivo calcium monitoring, dynamics of PDF projections, ArcLight, GCaMP6 imaging and sleep assays indicate that mir-92a suppresses neuronal excitability. In addition, mir-92a levels within PDF cells respond to light pulses and also affect the phase shift response. Translating ribosome affinity purification (TRAP) and in vitro luciferase reporter assay indicate that mir-92a suppresses expression of sirt2, which is homologous to human sir2 and sirt3. sirt2 RNAi also phenocopies mir-92a overexpression. These experiments indicate that sirt2 is a functional mir-92a target and that mir-92a modulates PDF neuronal excitability via suppressing SIRT2 levels in a rhythmic manner.

CLOCKWORK ORANGE Enhances PERIOD Mediated Rhythms in Transcriptional Repression by Antagonizing E-box Binding by CLOCK-CYCLE.

The Drosophila circadian oscillator controls daily rhythms in physiology, metabolism and behavior via transcriptional feedback loops. CLOCK-CYCLE (CLK-CYC) heterodimers initiate feedback loop function by binding E-box elements to activate per and tim transcription. PER-TIM heterodimers then accumulate, bind CLK-CYC to inhibit transcription, and are ultimately degraded to enable the next round of transcription. The timing of transcriptional events in this feedback loop coincide with, and are controlled by, rhythms in CLK-CYC binding to E-boxes. PER rhythmically binds CLK-CYC to initiate transcriptional repression, and subsequently promotes the removal of CLK-CYC from E-boxes. However, little is known about the mechanism by which CLK-CYC is removed from DNA. Previous studies demonstrated that the transcription repressor CLOCKWORK ORANGE (CWO) contributes to core feedback loop function by repressing per and tim transcription in cultured S2 cells and in flies. Here we show that CWO rhythmically binds E-boxes upstream of core clock genes in a reciprocal manner to CLK, thereby promoting PER-dependent removal of CLK-CYC from E-boxes, and maintaining repression until PER is degraded and CLK-CYC displaces CWO from E-boxes to initiate transcription. These results suggest a model in which CWO co-represses CLK-CYC transcriptional activity in conjunction with PER by competing for E-box binding once CLK-CYC-PER complexes have formed. Given that CWO orthologs DEC1 and DEC2 also target E-boxes bound by CLOCK-BMAL1, a similar mechanism may operate in the mammalian clock.

CRTC Potentiates Light-independent timeless Transcription to Sustain Circadian Rhythms in Drosophila.

Light is one of the strongest environmental time cues for entraining endogenous circadian rhythms. Emerging evidence indicates that CREB-regulated transcription co-activator 1 (CRTC1) is a key player in this pathway, stimulating light-induced Period1 (Per1) transcription in mammalian clocks. Here, we demonstrate a light-independent role of Drosophila CRTC in sustaining circadian behaviors. Genomic deletion of the crtc locus causes long but poor locomotor rhythms in constant darkness. Overexpression or RNA interference-mediated depletion of CRTC in circadian pacemaker neurons similarly impairs the free-running behavioral rhythms, implying that Drosophila clocks are sensitive to the dosage of CRTC. The crtc null mutation delays the overall phase of circadian gene expression yet it remarkably dampens light-independent oscillations of TIMELESS (TIM) proteins in the clock neurons. In fact, CRTC overexpression enhances CLOCK/CYCLE (CLK/CYC)-activated transcription from tim but not per promoter in clock-less S2 cells whereas CRTC depletion suppresses it. Consistently, TIM overexpression partially but significantly rescues the behavioral rhythms in crtc mutants. Taken together, our data suggest that CRTC is a novel co-activator for the CLK/CYC-activated tim transcription to coordinate molecular rhythms with circadian behaviors over a 24-hour time-scale. We thus propose that CRTC-dependent clock mechanisms have co-evolved with selective clock genes among different species.

Identification of Light-Sensitive Phosphorylation Sites on PERIOD That Regulate the Pace of Circadian Rhythms in Drosophila.

The main components regulating the pace of circadian (≅24 h) clocks in animals are PERIOD (PER) proteins, transcriptional regulators that undergo daily changes in levels and nuclear accumulation by means of complex multisite phosphorylation programs. In the present study, we investigated the function of two phosphorylation sites, at Ser826 and Ser828, located in a putative nuclear localization signal (NLS) on the Drosophila melanogaster PER protein. These sites are phosphorylated by DOUBLETIME (DBT; Drosophila homolog of CK1δ/ε), the key circadian kinase regulating the daily changes in PER stability and phosphorylation. Mutant flies in which phosphorylation at Ser826/Ser828 is blocked manifest behavioral rhythms with periods slightly longer than 1 h and with altered temperature compensation properties. Intriguingly, although phosphorylation at these sites does not influence PER stability, timing of nuclear entry, or transcriptional autoinhibition, the phospho-occupancy at Ser826/Ser828 is rapidly stimulated by light and blocked by TIMELESS (TIM), the major photosensitive clock component in Drosophila and a crucial binding partner of PER. Our findings identify the first phosphorylation sites on core clock proteins that are acutely regulated by photic cues and suggest that some phosphosites on PER proteins can modulate the pace of downstream behavioral rhythms without altering central aspects of the clock mechanism.

Drosophila circadian rhythms in seminatural environments: Summer afternoon component is not an artifact and requires TrpA1 channels

Under standard laboratory conditions of rectangular light/dark cycles and constant warm temperature, Drosophila melanogaster show bursts of morning (M) and evening (E) locomotor activity and a "siesta" in the middle of the day. These M and E components have been critical for developing the neuronal dual oscillator model in which clock gene expression in key cells generates the circadian phenotype. However, under natural European summer conditions of cycling temperature and light intensity, an additional prominent afternoon (A) component that replaces the siesta is observed. This component has been described as an "artifact" of the TriKinetics locomotor monitoring system that is used by many circadian laboratories world wide. Using video recordings, we show that the A component is not an artifact, neither in the glass tubes used in TriKinetics monitors nor in open-field arenas. By studying various mutants in the visual and peripheral and internal thermo-sensitive pathways, we reveal that the M component is predominantly dependent on visual input, whereas the A component requires the internal thermo-sensitive channel transient receptor potential A1 (TrpA1). Knockdown of TrpA1 in different neuronal groups reveals that the reported expression of TrpA1 in clock neurons is unlikely to be involved in generating the summer locomotor profile, suggesting that other TrpA1 neurons are responsible for the A component. Studies of circadian rhythms under seminatural conditions therefore provide additional insights into the molecular basis of circadian entrainment that would otherwise be lost under the usual standard laboratory protocols.

Clk post-transcriptional control denoises circadian transcription both temporally and spatially.

The transcription factor CLOCK (CLK) is essential for the development and maintenance of circadian rhythms in Drosophila. However, little is known about how CLK levels are controlled. Here we show that Clk mRNA is strongly regulated post-transcriptionally through its 3' UTR. Flies expressing Clk transgenes without normal 3' UTR exhibit variable CLK-driven transcription and circadian behaviour as well as ectopic expression of CLK-target genes in the brain. In these flies, the number of the key circadian neurons differs stochastically between individuals and within the two hemispheres of the same brain. Moreover, flies carrying Clk transgenes with deletions in the binding sites for the miRNA bantam have stochastic number of pacemaker neurons, suggesting that this miRNA mediates the deterministic expression of CLK. Overall our results demonstrate a key role of Clk post-transcriptional control in stabilizing circadian transcription, which is essential for proper development and maintenance of circadian rhythms in Drosophila.