Mammalian Circadian Clock Research Articles

Generalized michaelis-menten rate law beyond steady-state assumptions.

Despite over a century's use as a dominant paradigm in the description of biochemical rate processes, the Michaelis-Menten (MM) rate law stands on the restrictive assumption that the concentration of the complex of interacting molecules, at each moment, approaches an equilibrium much faster than the molecular concentration changes. The increasingly-appreciated, remedied form of the MM rate law is also based on this quasi-steady state assumption. Although this assumption may be valid for a range of biochemical systems, the exact extent of such systems is not clear. In this study, we relax the quasi-steady state requirement and propose the revised MM rate law for the interactions of molecules with active concentration changes over time. Our revised rate law, characterized by rigorously-derived time delay effects in molecular complex formation, improves the accuracy of models especially for protein-protein and protein-DNA interactions. Our simulation and empirical data analysis show that the improvement is not limited to the quantitatively better characterization of the dynamics, but also allows the prediction for qualitatively new patterns in the systems of interest. The latter include the oscillation condition and period patterns of the mammalian circadian clock and the spontaneous rhythmicity in the degradation rates of circadian proteins, both not properly captured by the previous approaches. Moreover, our revised rate law is capable of more accurate parameter estimation. This work offers an analytical framework for understanding rich dynamics of biomolecular systems, which goes beyond the quasi-steady state assumption.

Analysis of mammalian circadian clock protein complexes over a circadian cycle

Circadian rhythmicity is maintained by a set of core clock proteins including the transcriptional activators CLOCK and BMAL1, and the repressors PER (PER1, PER2, and PER3), CRY (CRY1 and CRY2), and CK1δ. In mice, peak expression of the repressors in the early morning reduces CLOCK- and BMAL1-mediated transcription/translation of the repressors themselves. By late afternoon the repressors are largely depleted by degradation, and thereby their expression is reactivated in a cycle repeated every 24h. Studies have characterized a variety of possible protein interactions and complexes associated with the function of this transcription-translation feedback loop. Our prior investigation suggested there were two circadian complexes responsible for rhythmicity, one containing CLOCK-BMAL and the other containing PER2, CRY1, and CK1δ. In this investigation, we acquired data from glycerol gradient centrifugation and gel filtration chromatography of mouse liver extracts obtained at different circadian times to further characterize circadian complexes. In addition, anti-PER2 and anti-CRY1 immunoprecipitates obtained from the same extracts were analyzed by liquid chromatography-tandem mass spectrometry to identify components of circadian complexes. Our results confirm the presence of discrete CLOCK-BMAL1 and PER-CRY-CK1δ complexes at the different circadian time points, provide masses of 255 and 707kDa, respectively, for these complexes, and indicate that these complexes are composed principally of the core circadian proteins.

Network-driven intracellular cAMP coordinates circadian rhythm in the suprachiasmatic nucleus.

The mammalian central circadian clock, located in the suprachiasmatic nucleus (SCN), coordinates the timing of physiology and behavior to local time cues. In the SCN, second messengers, such as cAMP and Ca2+, are suggested to be involved in the input and/or output of the molecular circadian clock. However, the functional roles of second messengers and their dynamics in the SCN remain largely unclear. In the present study, we visualized the spatiotemporal patterns of circadian rhythms of second messengers and neurotransmitter release in the SCN. Here, we show that neuronal activity regulates the rhythmic release of vasoactive intestinal peptides from the SCN, which drives the circadian rhythms of intracellular cAMP in the SCN. Furthermore, optical manipulation of intracellular cAMP levels in the SCN shifts molecular and behavioral circadian rhythms. Together, our study demonstrates that intracellular cAMP is a key molecule in the organization of the SCN circadian neuronal network.

Cryptochrome: An ancient blue light photoreceptor impacts modern mammalian physiology

Cryptochromes, evolutionally conserved and retained in mammals as transcriptional regulators having a repressive role in the transcription-translation feedback loop, the molecular mechanism behind the control of the endogenous mammalian circadian clock. This clock mechanism regulates the oscillation of a huge number of clock-controlled output genes. This in turn is responsible for modification of the physiological response of most organs and tissues, to coordinate with diurnal and seasonal changes in light and nutrient availability. Cryptochromes have also been found to participate in additional signalling cascades, outside of the circadian system, forming supplementary feedback loops that initiate cross-talk between systems influencing metabolism, inflammation and DNA damage response to maintain cellular homeostasis. This physiological organisation system has developed from Palaeolithic man but is still relevant in our modern world.

Level of constitutively expressed BMAL1 affects the robustness of circadian oscillations

The circadian rhythm is a biological oscillation of physiological activities with a period of approximately 24 h, that is driven by a cell-autonomous oscillator called the circadian clock. The current model of the mammalian circadian clock is based on a transcriptional-translational negative feedback loop in which the protein products of clock genes accumulate in a circadian manner and repress their own transcription. However, several studies have revealed that constitutively expressed clock genes can maintain circadian oscillations. To understand the underlying mechanism, we expressed Bmal1 in Bmal1-disrupted cells using a doxycycline-inducible promoter and monitored Bmal1 and Per2 promoter activity using luciferase reporters. Although the levels of BMAL1 and other clock proteins, REV-ERBα and CLOCK, showed no obvious rhythmicity, robust circadian oscillation in Bmal1 and Per2 promoter activities with the correct phase relationship was observed, which proceeded in a doxycycline-concentration-dependent manner. We applied transient response analysis to the Bmal1 promoter activity in the presence of various doxycycline concentrations. Based on the obtained transfer functions, we suggest that, at least in our experimental system, BMAL1 is not directly involved in the oscillatory process, but modulates the oscillation robustness by regulating basal clock gene promoter activity.

Discovery of a small molecule that selectively destabilizes Cryptochrome 1 and enhances life span in p53 knockout mice

Cryptochromes are negative transcriptional regulators of the circadian clock in mammals. It is not clear how reducing the level of endogenous CRY1 in mammals will affect circadian rhythm and the relation of such a decrease with apoptosis. Here, we discovered a molecule (M47) that destabilizes Cryptochrome 1 (CRY1) both in vitro and in vivo. The M47 selectively enhanced the degradation rate of CRY1 by increasing its ubiquitination and resulted in increasing the circadian period length of U2OS Bmal1-dLuc cells. In addition, subcellular fractionation studies from mice liver indicated that M47 increased degradation of the CRY1 in the nucleus. Furthermore, M47-mediated CRY1 reduction enhanced oxaliplatin-induced apoptosis in Ras-transformed p53 null fibroblast cells. Systemic repetitive administration of M47 increased the median lifespan of p53−/− mice by ~25%. Collectively our data suggest that M47 is a promising molecule to treat forms of cancer depending on the p53 mutation.

Developmental light deprivation transiently reduces the expression of vGluT2 and GluN2B in the rat ventral suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the most important circadian clock in mammals. The SCN synchronizes to environmental light via the retinohypothalamic tract (RHT), which is an axon cluster derived from melanopsin-expressing intrinsic photosensitive retinal ganglion cells. Investigations on the development of the nonimage-forming pathway and the RHT are scarce. Previous studies imply that light stimulation during postnatal development is not needed to make the RHT functional at adult stages. Here, we examined the effects of light deprivation (i.e., constant darkness (DD) rearing) during postnatal development on the expression in the ventral SCN of two crucial proteins for the synchronization of circadian rhythms to light: the presynaptic vesicular glutamate transporter type 2 (vGluT2) and the GluN2B subunit of the postsynaptic NMDA receptor. We found that animals submitted to DD conditions exhibited a transitory reduction in the expression of vGluT2 (at P12-19) and of GluN2B (at P7-9) that was compensated at older stages. These findings support the hypothesis that visual stimulation during early ages is not decisive for normal development of the RHT-SCN pathway.

Nmu/Nms/Gpr176 Triple-Deficient Mice Show Enhanced Light-Resetting of Circadian Locomotor Activity.

The suprachiasmatic nucleus (SCN) is the master circadian clock in mammals and is properly entrained by environmental light cycle. However, the molecular mechanism(s) determining the magnitude of phase shift by light is still not fully understood. The orphan G-protein-coupled receptor Gpr176 is enriched in the SCN, controls the pace (period) of the circadian rhythm in behavior but is not apparently involved in the light entrainment; Gpr176-/- animals display a shortened circadian period in constant darkness but their phase-resetting responses to light are normal. Here, we performed microarray analysis and identified enhanced mRNA expression of neuromedin U (Nmu) and neuromedin S (Nms) in the SCN of Gpr176-/- mice. By generating C57BL/6J-backcrossed Nmu/Nms/Gpr176 triple knockout mice, we noted that the mutant mice had a greater magnitude of phase shift in response to early subjective night light than wildtype mice, while Nmu/Nms double knockout mice as well as Gpr176 knockout mice are normal in the phase shifts induced by light. At the molecular level, Nmu-/-Nms-/-Gpr176-/- mice had a reduced induction of Per1 and cFos mRNA expression in the SCN by light and mildly upregulated circadian expression of Per2, Prok2, Rgs16, and Rasl11b. These expressional changes may underlie the phenotype of the Nmu/Nms/Gpr176 knockout mice. Our data argue that there is a mechanism requiring Nmu, Nms, and Gpr176 for the proper modulation of light-induced phase shift in mice. Simultaneous modulation of Nmu/Nms/Gpr176 may provide a potential target option for modulating the circadian clock.

Decoupling PER phosphorylation, stability and rhythmic expression from circadian clock function by abolishing PER-CK1 interaction

Robust rhythms of abundances and phosphorylation profiles of PERIOD proteins were thought be the master rhythms that drive mammalian circadian clock functions. PER stability was proposed to be a major determinant of period length. In mammals, CK1 forms stable complexes with PER. Here we identify the PER residues essential for PER-CK1 interaction. In cells and in mice, their mutation abolishes PER phosphorylation and CLOCK hyperphosphorylation, resulting in PER stabilization, arrhythmic PER abundance and impaired negative feedback process, indicating that PER acts as the CK1 scaffold in circadian feedback mechanism. Surprisingly, the mutant mice exhibit robust short period locomotor activity and other physiological rhythms but low amplitude molecular rhythms. PER-CK1 interaction has two opposing roles in regulating CLOCK-BMAL1 activity. These results indicate that the circadian clock can function independently of PER phosphorylation and abundance rhythms due to another PER-CRY-dependent feedback mechanism and that period length can be uncoupled from PER stability.

CNOT1 regulates circadian behaviour through Per2 mRNA decay in a deadenylation-dependent manner

ABSTRACT Circadian clocks are an endogenous internal timekeeping mechanism that drives the rhythmic expression of genes, controlling the 24 h oscillatory pattern in behaviour and physiology. It has been recently shown that post-transcriptional mechanisms are essential for controlling rhythmic gene expression. Controlling the stability of mRNA through poly(A) tail length modulation is one such mechanism. In this study, we show that Cnot1, encoding the scaffold protein of the CCR4-NOT deadenylase complex, is highly expressed in the suprachiasmatic nucleus, the master timekeeper. CNOT1 deficiency in mice results in circadian period lengthening and alterations in the mRNA and protein expression patterns of various clock genes, mainly Per2. Per2 mRNA exhibited a longer poly(A) tail and increased mRNA stability in Cnot1+/− mice. CNOT1 is recruited to Per2 mRNA through BRF1 (ZFP36L1), which itself oscillates in antiphase with Per2 mRNA. Upon Brf1 knockdown, Per2 mRNA is stabilized leading to increased PER2 expression levels. This suggests that CNOT1 plays a role in tuning and regulating the mammalian circadian clock.

Cell Type-Specific Genetic Manipulation and Impaired Circadian Rhythms in Vip tTA Knock-In Mice.

The suprachiasmatic nucleus (SCN), the central circadian clock in mammals, is a neural network consisting of various types of GABAergic neurons, which can be differentiated by the co-expression of specific peptides such as vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP). VIP has been considered as a critical factor for the circadian rhythmicity and synchronization of individual SCN neurons. However, the precise mechanisms of how VIP neurons regulate SCN circuits remain incompletely understood. Here, we generated Vip tTA knock-in mice that express tetracycline transactivator (tTA) specifically in VIP neurons by inserting tTA sequence at the start codon of Vip gene. The specific and efficient expression of tTA in VIP neurons was verified using EGFP reporter mice. In addition, combined with Avp-Cre mice, Vip tTA mice enabled us to simultaneously apply different genetic manipulations to VIP and AVP neurons in the SCN. Immunostaining showed that VIP is expressed at a slightly reduced level in heterozygous Vip tTA mice but is completely absent in homozygous mice. Consistently, homozygous Vip tTA mice showed impaired circadian behavioral rhythms similar to those of Vip knockout mice, such as attenuated rhythmicity and shortened circadian period. In contrast, heterozygous mice demonstrated normal circadian behavioral rhythms comparable to wild-type mice. These data suggest that Vip tTA mice are a valuable genetic tool to express exogenous genes specifically in VIP neurons in both normal and VIP-deficient mice, facilitating the study of VIP neuronal roles in the SCN neural network.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Combined multiple transcriptional repression mechanisms generate ultrasensitivity and oscillations.

Transcriptional repression can occur via various mechanisms, such as blocking, sequestration and displacement. For instance, the repressors can hold the activators to prevent binding with DNA or can bind to the DNA-bound activators to block their transcriptional activity. Although the transcription can be completely suppressed with a single mechanism, multiple repression mechanisms are used together to inhibit transcriptional activators in many systems, such as circadian clocks and NF-κB oscillators. This raises the question of what advantages arise if seemingly redundant repression mechanisms are combined. Here, by deriving equations describing the multiple repression mechanisms, we find that their combination can synergistically generate a sharply ultrasensitive transcription response and thus strong oscillations. This rationalizes why the multiple repression mechanisms are used together in various biological oscillators. The critical role of such combined transcriptional repression for strong oscillations is further supported by our analysis of formerly identified mutations disrupting the transcriptional repression of the mammalian circadian clock. The hitherto unrecognized source of the ultrasensitivity, the combined transcriptional repressions, can lead to robust synthetic oscillators with a previously unachievable simple design.

Transcriptional Feedback Loops in the Caprine Circadian Clock System.

The circadian clock system is based on interlocked positive and negative transcriptional and translational feedback loops of core clock genes and their encoded proteins. The mammalian circadian clock system has been extensively investigated using mouse models, but has been poorly investigated in diurnal ruminants. In this study, goat embryonic fibroblasts (GEFs) were isolated and used as a cell model to elucidate the caprine circadian clock system. Real-time quantitative PCR analysis showed that several clock genes and clock-controlled genes were rhythmically expressed in GEFs over a 24 h period after dexamethasone stimulation. Immunofluorescence revealed that gBMAL1 and gNR1D1 proteins were expressed in GEFs, and western blotting analysis further verified that the proteins were expressed with circadian rhythmic changes. Diurnal changes in clock and clock-controlled gene expression at the mRNA and protein levels were also observed in goat liver and kidney tissues at two representative time points in vivo. Amino acid sequences and tertiary structures of goat BMAL1 and CLOCK proteins were found to be highly homologous to those in mice and humans. In addition, a set of goat representative clock gene orthologs and the promoter regions of two clock genes of goats and mice were cloned. Dual-luciferase reporter assays showed that gRORα could activate the promoter activity of the goat BMAL1, while gNR1D1 repressed it. The elevated pGL4.10-gNR1D1-Promoter-driven luciferase activity induced by mBMAL1/mCLOCK was much higher than that induced by gBMAL1/gCLOCK, and the addition of gCRY2 or mPER2 repressed it. Real-time bioluminescence assays revealed that the transcriptional activity of BMAL1 and NR1D1 in goats and mice exhibited rhythmic changes over a period of approximately 24 h in NIH3T3 cells or GEFs. Notably, the amplitudes of gBMAL1 and gNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells were higher than those in GEFs, while mBMAL1 and mNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells had the highest amplitude. In sum, transcriptional and translational loops of the mammalian circadian clock system were found to be broadly conserved in goats and not as robust as those found in mice, at least in the current experimental models. Further studies are warranted to elucidate the specific molecular mechanisms involved.

The role of cell-autonomous circadian oscillation of Cry transcription in circadian rhythm generation.

The current model of the mammalian circadian clock describes cell-autonomous and negative feedback-driven circadian oscillation of Cry and Per transcription as the core circadian rhythm generator. However, the actual contribution of this oscillation to circadian rhythm generation remains undefined. Here we perform targeted disruption of cis elements indispensable for cell-autonomous Cry oscillation. Mice lacking overt cell-autonomous Cry oscillation show robust circadian rhythms in locomotor activity. In addition, tissue-autonomous circadian rhythms are robust in the absence of overt Cry oscillation. Unexpectedly, although the absence of overt Cry oscillation leads to severe attenuation of Per oscillation at the cell-autonomous level, circadian rhythms in Per2 accumulation remain robust. As a mechanism to explain this counterintuitive result, Per2 half-life shows cell-autonomous circadian rhythms independent of Cry and Per oscillation. The cell-autonomous circadian clock may therefore remain partially functional even in the absence of overt Cry and Per oscillation because of circadian oscillation in Per2 degradation.

Promoter methylation in a mixed feedback loop circadian clock model.

We investigate how epigenetic modifications to clock gene promoters affect transcriptomic activity in the circadian clock. Motivated by experimental observations that link DNA methylation with the behavior of the clock, we introduce and analyze an extension of the mixed feedback loop (MFL) model of François and Hakim. We extend the original model to include an additional methylated promoter state and allow for reversible protein sequestration, an important feature for circadian applications. First, working with the general form of the MFL model, we find that the qualitative behavior of the model is dictated by the promoter state with the highest transcription rate. We then build on the work of Kim and Forger, who analyzed the stability of the mammalian circadian clock by using a reduced form of the MFL model. We present a rigorous procedure for translating between the MFL model and the reduction of Kim and Forger. We then propose a model reduction more appropriate for the study of oscillatory promoter states, making use of a fully coupled quasi-steady-state approximation rather than the standard partially uncoupled quasi-steady-state approach. Working with the novel reduced form of the model, we find substantial differences in the transcription function and show that, although methylation contributes to period control, excessive methylation can abolish rhythmicity. Altogether our results show that even in a minimal clock model, DNA methylation has a nontrivial influence on the system's ability to oscillate.

Mathematical analysis of robustness of oscillations in models of the mammalian circadian clock.

Circadian rhythms in a wide range of organisms are mediated by molecular mechanisms based on transcription-translation feedback. In this paper, we use bifurcation theory to explore mathematical models of genetic oscillators, based on Kim & Forger’s interpretation of the circadian clock in mammals. At the core of their models is a negative feedback loop whereby PER proteins (PER1 and PER2) bind to and inhibit their transcriptional activator, BMAL1. For oscillations to occur, the dissociation constant of the PER:BMAL1 complex, , must be ≤ 0.04 nM, which is orders of magnitude smaller than a reasonable expectation of 1–10 nM for this protein complex. We relax this constraint by two modifications to Kim & Forger’s ‘single negative feedback’ (SNF) model: first, by introducing a multistep reaction chain for posttranscriptional modifications of Per mRNA and posttranslational phosphorylations of PER, and second, by replacing the first-order rate law for degradation of PER in the nucleus by a Michaelis-Menten rate law. These modifications increase the maximum allowable to ~2 nM. In a third modification, we consider an alternative rate law for gene transcription to resolve an unrealistically large rate of Per2 transcription at very low concentrations of BMAL1. Additionally, we studied extensions of the SNF model to include a second negative feedback loop (involving REV-ERB) and a supplementary positive feedback loop (involving ROR). Contrary to Kim & Forger’s observations of these extended models, we find that, with our modifications, the supplementary positive feedback loop makes the oscillations more robust than observed in the models with one or two negative feedback loops. However, all three models are similarly robust when accounting for circadian rhythms (~24 h period) with ≥ 1 nM. Our results provide testable predictions for future experimental studies.

Polygalae Radix shortens the circadian period through activation of the CaMKII pathway

Context The mammalian circadian clock system regulates physiological function. Crude drugs, containing Polygalae Radix, and Kampō, combining multiple crude drugs, have been used to treat various diseases, but few studies have focussed on the circadian clock. Objective We examine effective crude drugs, which cover at least one or two of Kampō, for the shortening effects on period length of clock gene expression rhythm, and reveal the mechanism of shortening effects. Materials and methods We prepared 40 crude drugs. In the in vitro experiments, we used mouse embryonic fibroblasts from PERIOD2::LUCIFERASE knock-in mice (background; C57BL/6J mice) to evaluate the effect of crude drugs on the period length of core clock gene, Per2, expression rhythm by chronic treatment (six days) with distilled water or crude drugs (100 μg/mL). In the in vivo experiments, we evaluated the free-running period length of C57BL/6J mice fed AIN-93M or AIN-93M supplemented with 1% crude drug (6 weeks) that shortened the period length of the PERIOD2::LUCIFERASE expression rhythm in the in vitro experiments. Results We found that Polygalae Radix (ED50: 24.01 μg/mL) had the most shortened PERIOD2::LUCIFERASE rhythm period length in 40 crude drugs and that the CaMKII pathway was involved in this effect. Moreover, long-term feeding with AIN-93M+Polygalae Radix slightly shortened the free-running period of the mouse locomotor activity rhythm. Discussion and conclusions Our results indicate that Polygalae Radix may be regarded as a new therapy for circadian rhythm disorder and that the CaMKII pathway may be regarded as a target pathway for circadian rhythm disorders.

Quantification of protein abundance and interaction defines a mechanism for operation of the circadian clock.

The mammalian circadian clock exerts control of daily gene expression through cycles of DNA binding. Here, we develop a quantitative model of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We used quantitative imaging to track dynamic changes in endogenous labelled proteins across peripheral tissues and the SCN. We determine the contribution of multiple rhythmic processes coordinating BMAL1 DNA binding, including cycling molecular abundance, binding affinities, and repression. We find nuclear BMAL1 concentration determines corresponding CLOCK through heterodimerisation and define a DNA residence time of this complex. Repression of CLOCK:BMAL1 is achieved through rhythmic changes to BMAL1:CRY1 association and high-affinity interactions between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.

A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System.

The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day–night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator’s period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep–wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans.