Pleiotropic brain function of whirlin identified by a novel mutation.

Despite some evidence indicating diverse roles of whirlin in neurons, the functional corollary of whirlin gene function and behavior has not been investigated or broadly characterized. A single nucleotide variant was identified from our recessive ENU-mutagenesis screen at a donor-splice site in whirlin, a protein critical for proper sensorineural hearing function. The mutation (head-bob, hb) led to partial intron-retention causing a frameshift and introducing a premature termination codon. Mutant mice had a head-bobbing phenotype and significant hyperactivity across several phenotyping tests. Lack of complementation of head-bob with whirler mutant mice confirmed the head-bob mutation as functionally distinct with compound mutants having a mild-moderate hearing defect. Utilizing transgenics, we demonstrate rescue of the hyperactive phenotype and combined with the expression profiling data conclude whirlin plays an essential role in activity-related behaviors. These results highlight a pleiotropic role of whirlin within the brain and implicate alternative, central mediated pathways in its function.

A missense mutation in zinc finger homeobox-3 (ZFHX3) impedes growth and alters metabolism and hypothalamic gene expression in mice.

A protein altering variant in the gene encoding zinc finger homeobox-3 (ZFHX3) has recently been associated with lower BMI in a human genome-wide association study. We investigated metabolic parameters in mice harboring a missense mutation in Zfhx3 (Zfhx3Sci/+ ) and looked for altered in situ expression of transcripts that are associated with energy balance in the hypothalamus to understand how ZFHX3 may influence growth and metabolic effects. One-year-old male and female Zfhx3Sci/+ mice weighed less, had shorter body length, lower fat mass, smaller mesenteric fat depots, and lower circulating insulin, leptin, and insulin-like growth factor-1 (IGF1) concentrations than Zfhx3+/+ littermates. In a second cohort of 9-20-week-old males and females, Zfhx3Sci/+ mice ate less than wildtype controls, in proportion to body weight. In a third cohort of female-only Zfhx3Sci/+ and Zfhx3+/+ mice that underwent metabolic phenotyping from 6 to 14 weeks old, Zfhx3Sci/+ mice weighed less and had lower lean mass and energy expenditure, but fat mass did not differ. We detected increased expression of somatostatin and decreased expression of growth hormone-releasing hormone and growth hormone-receptor mRNAs in the arcuate nucleus (ARC). Similarly, ARC expression of orexigenic neuropeptide Y was decreased and ventricular ependymal expression of orphan G protein-coupled receptor Gpr50 was decreased. We demonstrate for the first time an energy balance effect of the Zfhx3Sci mutation, likely by altering expression of key ARC neuropeptides to alter growth, food intake, and energy expenditure.

Harvesting mouse suprachiasmatic nucleus by vibrating microtome for diurnal transcriptome analysis.

The mammalian suprachiasmatic nucleus (SCN) is the principal circadian clock that synchronizes daily behavioral and physiological responses in response to environmental cues. Here, we present a protocol for harvesting mouse SCN by vibrating microtome for diurnal transcriptome analysis. We describe steps for mouse entrainment, isolation of the SCN, tissue preparation, slicing with a vibratome, and handling of the harvested SCN for RNA extraction. This protocol can also be used for harvesting other mammalian brain regions for genomic studies.

DNA methylation networks underlying mammalian traits.

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

Dynamic modulation of genomic enhancer elements in the suprachiasmatic nucleus, the site of the mammalian circadian clock.

The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, synchronizes and maintains daily cellular and physiological rhythms across the body, in accordance with environmental and visceral cues. Consequently, the systematic regulation of spatiotemporal gene transcription in the SCN is vital for daily timekeeping. So far, the regulatory elements assisting circadian gene transcription have only been studied in peripheral tissues, lacking the critical neuronal dimension intrinsic to the role of the SCN as central brain pacemaker. By using histone-ChIP-seq, we identified SCN-enriched gene regulatory elements that associated with temporal gene expression. Based on tissue-specific H3K27ac and H3K4me3 marks, we successfully produced the first-ever SCN gene-regulatory map. We found that a large majority of SCN enhancers not only show robust 24-h rhythmic modulation in H3K27ac occupancy, peaking at distinct times of day, but also possess canonical E-box (CACGTG) motifs potentially influencing downstream cycling gene expression. To establish enhancer-gene relationships in the SCN, we conducted directional RNA-seq at six distinct times across the day and night, and studied the association between dynamically changing histone acetylation and gene transcript levels. About 35% of the cycling H3K27ac sites were found adjacent to rhythmic gene transcripts, often preceding the rise in mRNA levels. We also noted that enhancers encompass noncoding, actively transcribing enhancer RNAs (eRNAs) in the SCN, which in turn oscillate, along with cyclic histone acetylation, and correlate with rhythmic gene transcription. Taken together, these findings shed light on genome-wide pretranscriptional regulation operative in the central clock that confers its precise and robust oscillation necessary to orchestrate daily timekeeping in mammals.

Shift work-like patterns effect on female and male mouse behavior.

Shift work (work outside of standard daylight hours) is common throughout the Western world. However, there are notable health consequences to shift work, including increased prevalence of mental health and sleep disorders in shift worker populations. Therefore, the health and wellbeing of shift workers is a public health concern that needs to be addressed. Here we investigate the effects of two separate light induced shift work-like patterns on male and female mouse behaviour (anxiety-like, exploration, marble burying, startle reflex and circadian rhythms). After 6 weeks of shift-like disruptions patterns, animals displayed no behavioral differences in exploration, marble burying and startle reflex. Interestingly however, we identified sex specific and disruption specific effects in light aversion and wheel running activities. Notably, analysis of the activity patterns of animals in disruptive conditions demonstrated that they maintained a degree of rhythmicity through the disruption period, which may explain the lack of behavioral differences in most behavioral tests.

Chronic Exposure to Dim Light at Night or Irregular Lighting Conditions Impact Circadian Behavior, Motor Coordination, and Neuronal Morphology.

Mistimed exposure to light has been demonstrated to negatively affect multiple aspects of physiology and behavior. Here we analyzed the effects of chronic exposure to abnormal lighting conditions in mice. We exposed mice for 1 year to either: a standard light/dark cycle, a "light-pollution" condition in which low levels of light were present in the dark phase of the circadian cycle (dim light at night, DLAN), or altered light cycles in which the length of the weekday and weekend light phase differed by 6 h ("social jetlag"). Mice exhibited several circadian activity phenotypes, as well as changes in motor function, associated particularly with the DLAN condition. Our data suggest that these phenotypes might be due to changes outside the core clock. Dendritic spine changes in other brain regions raise the possibility that these phenotypes are mediated by changes in neuronal coordination outside of the clock. Given the prevalence of artificial light exposure in the modern world, further work is required to establish whether these negative effects are observed in humans as well.

Tissue Proteome of 2-Hydroxyacyl-CoA Lyase Deficient Mice Reveals Peroxisome Proliferation and Activation of ω-Oxidation.

Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of ß-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1-/- mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1-/- liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1-/- mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1-/- mice in better understanding disease mechanism in fatty acid α-oxidation disorders.

Zfhx3-mediated genetic ablation of the SCN abolishes light entrainable circadian activity while sparing food anticipatory activity.

Circadian rhythms persist in almost all organisms and are crucial for maintaining appropriate timing in physiology and behaviour. Here, we describe a mouse mutant where the central mammalian pacemaker, the suprachiasmatic nucleus (SCN), has been genetically ablated by conditional deletion of the transcription factor Zfhx3 in the developing hypothalamus. Mutants were arrhythmic over the light-dark cycle and in constant darkness. Moreover, rhythms of metabolic parameters were ablated in vivo although molecular oscillations in the liver maintained some rhythmicity. Despite disruptions to SCN cell identity and circuitry, mutants could still anticipate food availability, yet other zeitgebers - including social cues from cage-mates - were ineffective in restoring rhythmicity although activity levels in mutants were altered. This work highlights a critical role for Zfhx3 in the development of a functional SCN, while its genetic ablation further defines the contribution of SCN circuitry in orchestrating physiological and behavioral responses to environmental signals.

Comprehensive phenotypic analysis of the Dp1Tyb mouse strain reveals a broad range of Down syndrome-related phenotypes.

Down syndrome (DS), trisomy 21, results in many complex phenotypes including cognitive deficits, heart defects and craniofacial alterations. Phenotypes arise from an extra copy of human chromosome 21 (Hsa21) genes. However, these dosage-sensitive causative genes remain unknown. Animal models enable identification of genes and pathological mechanisms. The Dp1Tyb mouse model of DS has an extra copy of 63% of Hsa21-orthologous mouse genes. In order to establish whether this model recapitulates DS phenotypes, we comprehensively phenotyped Dp1Tyb mice using 28 tests of different physiological systems and found that 468 out of 1800 parameters were significantly altered. We show that Dp1Tyb mice have wide-ranging DS-like phenotypes, including aberrant erythropoiesis and megakaryopoiesis, reduced bone density, craniofacial changes, altered cardiac function, a pre-diabetic state, and deficits in memory, locomotion, hearing and sleep. Thus, Dp1Tyb mice are an excellent model for investigating complex DS phenotype-genotype relationships for this common disorder.

Zfhx3 modulates retinal sensitivity and circadian responses to light.

Mutations in transcription factors often exhibit pleiotropic effects related to their complex expression patterns and multiple regulatory targets. One such mutation in the zinc finger homeobox 3 (ZFHX3) transcription factor, short circuit (Sci, Zfhx3Sci/+ ), is associated with significant circadian deficits in mice. However, given evidence of its retinal expression, we set out to establish the effects of the mutation on retinal function using molecular, cellular, behavioral and electrophysiological measures. Immunohistochemistry confirms the expression of ZFHX3 in multiple retinal cell types, including GABAergic amacrine cells and retinal ganglion cells including intrinsically photosensitive retinal ganglion cells (ipRGCs). Zfhx3Sci/+ mutants display reduced light responsiveness in locomotor activity and circadian entrainment, relatively normal electroretinogram and optomotor responses but exhibit an unexpected pupillary reflex phenotype with markedly increased sensitivity. Furthermore, multiple electrode array recordings of Zfhx3Sci/+ retina show an increased sensitivity of ipRGC light responses.

Simultaneous Assessment of Circadian Rhythms and Sleep in Mice Using Passive Infrared Sensors: A User's Guide.

The 24-hr cycle of activity and sleep provides perhaps the most familiar example of circadian rhythms. In mammals, circadian activity rhythms are generated by a master biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). This clock is synchronized (entrained) to the external light environment via light input from retinal photoreceptors. However, sleep is not a simple circadian output and also is regulated by a homeostatic process whereby extended wakefulness increases the need for subsequent sleep. As such, the amount and distribution of sleep depends upon the interaction between both circadian and homeostatic processes. Moreover, the study of circadian activity and sleep is not confined only to these specialized fields. Sleep and circadian rhythm disruption is common in many conditions, ranging from neurological and metabolic disorders to aging. Such disruption is associated with a range of negative consequences including cognitive impairment and mood disorders, as well as immune and metabolic dysfunction. As circadian activity and sleep are hallmarks of normal healthy physiology, they also provide valuable welfare indicators. However, traditional methods for the monitoring of circadian rhythms and sleep in mice can require separate specialized resources as well as significant expertise. Here, we outline a low-cost, non-invasive, and open-source method for the simultaneous assessment of circadian activity and sleep in mice. This protocol describes both the assembly of the hardware used and the capture and analysis of data without the need for expertise in electronics or data processing. © 2020 Wiley Periodicals LLC. Basic Protocol: Assembly of a PIR system for basic activity and sleep recordings Alternate Protocol: Data collection using Raspberry Pi Support Protocol: Circadian analysis using PIR sensors.

Forward genetics identifies a novel sleep mutant with sleep state inertia and REM sleep deficits.

Switches between global sleep and wakefulness states are believed to be dictated by top-down influences arising from subcortical nuclei. Using forward genetics and in vivo electrophysiology, we identified a recessive mouse mutant line characterized by a substantially reduced propensity to transition between wake and sleep states with an especially pronounced deficit in initiating rapid eye movement (REM) sleep episodes. The causative mutation, an Ile102Asn substitution in the synaptic vesicular protein, VAMP2, was associated with morphological synaptic changes and specific behavioral deficits, while in vitro electrophysiological investigations with fluorescence imaging revealed a markedly diminished probability of vesicular release in mutants. Our data show that global shifts in the synaptic efficiency across brain-wide networks leads to an altered probability of vigilance state transitions, possibly as a result of an altered excitability balance within local circuits controlling sleep-wake architecture.

The guanine nucleotide exchange factor, Spata13, influences social behaviour and nocturnal activity.

Spermatogenesis-associated protein 13 (Spata13) is a guanine nucleotide exchange factor (GEF) enriched in discrete brain regions in the adult, with pronounced expression in the extended central amygdala (CeA). Loss of Spata13, also known as the adenomatous polyposis coli exchange factor Asef2, has no identifiable phenotype although it has been shown to reduce the number and size of intestinal tumours in Apc (Min/+) mice. Nevertheless, its brain-related functions have not been investigated. To pursue this, we have generated a Spata13 knockout mouse line using CRISPR-mediated deletion of an exon containing the GTPase domain that is common to multiple isoforms. Homozygous mutants were viable and appeared normal. We subjected both male and female cohorts to a comprehensive battery of behavioural tests designed to investigate particular CeA-related functions. Here, we show that Spata13 modulates social behaviour with homozygous mutants being subordinate to wildtype controls. Furthermore, female homozygotes show increased activity in home cages during the dark phase of the light-dark cycle. In summary, Spata13 modulates social hierarchy in both male and female mice in addition to affecting voluntary activity in females.

Loss of Frrs1l disrupts synaptic AMPA receptor function, and results in neurodevelopmental, motor, cognitive and electrographical abnormalities.

Loss-of-function mutations in a human AMPA receptor-associated protein, ferric chelate reductase 1-like (FRRS1L), are associated with a devastating neurological condition incorporating choreoathetosis, cognitive deficits and epileptic encephalopathies. Furthermore, evidence from overexpression and ex vivo studies has implicated FRRS1L in AMPA receptor biogenesis, suggesting that changes in glutamatergic signalling might underlie the disorder. Here, we investigated the neurological and neurobehavioural correlates of the disorder using a mouse Frrs1l null mutant. The study revealed several neurological defects that mirrored those seen in human patients. We established that mice lacking Frrs1l suffered from a broad spectrum of early-onset motor deficits with no progressive, age-related deterioration. Moreover, Frrs1l-/- mice were hyperactive, irrespective of test environment, exhibited working memory deficits and displayed significant sleep fragmentation. Longitudinal electroencephalographic (EEG) recordings also revealed abnormal EEG results in Frrs1l-/- mice. Parallel investigations into disease aetiology identified a specific deficiency in AMPA receptor levels in the brain of Frrs1l-/- mice, while the general levels of several other synaptic components remained unchanged, with no obvious alterations in the number of synapses. Furthermore, we established that Frrsl1 deletion results in an increased proportion of immature AMPA receptors, indicated by incomplete glycosylation of GLUA2 (also known as GRIA2) and GLUA4 (also known as GRIA4) AMPA receptor proteins. This incomplete maturation leads to cytoplasmic retention and a reduction of those specific AMPA receptor levels in the postsynaptic membrane. Overall, this study determines, for the first time in vivo, how loss of FRRS1L function can affect glutamatergic signalling, and provides mechanistic insight into the development and progression of a human hyperkinetic disorder.This article has an associated First Person interview with the first author of the paper.

Modelling physical resilience in ageing mice.

Geroprotectors, a class of drugs targeting multiple deficits occurring with age, necessitate the development of new animal models to test their efficacy. The COST Action MouseAGE is a European network whose aim is to reach consensus on the translational path required for geroprotectors, interventions targeting the biology of ageing. In our previous work we identified frailty and loss of resilience as a potential target for geroprotectors. Frailty is the result of an accumulation of deficits, which occurs with age and reduces the ability to respond to adverse events (physical resilience). Modelling frailty and physical resilience in mice is challenging for many reasons. There is no consensus on the precise definition of frailty and resilience in patients or on how best to measure it. This makes it difficult to evaluate available mouse models. In addition, the characterization of those models is poor. Here we review potential models of physical resilience, focusing on those where there is some evidence that the administration of acute stressors requires integrative responses involving multiple tissues and where aged mice showed a delayed recovery or a worse outcome then young mice in response to the stressor. These models include sepsis, trauma, drug- and radiation exposure, kidney and brain ischemia, exposure to noise, heat and cold shock.

Differential roles for cryptochromes in the mammalian retinal clock.

Cryptochromes 1 and 2 (CRY1/2) are key components of the negative limb of the mammalian circadian clock. Like many peripheral tissues, Cry1 and -2 are expressed in the retina, where they are thought to play a role in regulating rhythmic physiology. However, studies differ in consensus as to their localization and function, and CRY1 immunostaining has not been convincingly demonstrated in the retina. Here we describe the expression and function of CRY1 and -2 in the mouse retina in both sexes. Unexpectedly, we show that CRY1 is expressed throughout all retinal layers, whereas CRY2 is restricted to the photoreceptor layer. Retinal period 2::luciferase recordings from CRY1-deficient mice show reduced clock robustness and stability, while those from CRY2-deficient mice show normal, albeit long-period, rhythms. In functional studies, we then investigated well-defined rhythms in retinal physiology. Rhythms in the photopic electroretinogram, contrast sensitivity, and pupillary light response were all severely attenuated or abolished in CRY1-deficient mice. In contrast, these physiological rhythms are largely unaffected in mice lacking CRY2, and only photopic electroretinogram rhythms are affected. Together, our data suggest that CRY1 is an essential component of the mammalian retinal clock, whereas CRY2 has a more limited role.-Wong, J. C. Y., Smyllie, N. J., Banks, G. T., Pothecary, C. A., Barnard, A. R., Maywood, E. S., Jagannath, A., Hughes, S., van der Horst, G. T. J., MacLaren, R. E., Hankins, M. W., Hastings, M. H., Nolan, P. M., Foster, R. G., Peirson, S. N. Differential roles for cryptochromes in the mammalian retinal clock.

Assessing mouse behaviour throughout the light/dark cycle using automated in-cage analysis tools.

An important factor in reducing variability in mouse test outcomes has been to develop assays that can be used for continuous automated home cage assessment. Our experience has shown that this has been most evidenced in long-term assessment of wheel-running activity in mice. Historically, wheel-running in mice and other rodents have been used as a robust assay to determine, with precision, the inherent period of circadian rhythms in mice. Furthermore, this assay has been instrumental in dissecting the molecular genetic basis of mammalian circadian rhythms. In teasing out the elements of this test that have determined its robustness - automated assessment of an unforced behaviour in the home cage over long time intervals - we and others have been investigating whether similar test apparatus could be used to accurately discriminate differences in distinct behavioural parameters in mice. Firstly, using these systems, we explored behaviours in a number of mouse inbred strains to determine whether we could extract biologically meaningful differences. Secondly, we tested a number of relevant mutant lines to determine how discriminative these parameters were. Our findings show that, when compared to conventional out-of-cage phenotyping, a far deeper understanding of mouse mutant phenotype can be established by monitoring behaviour in the home cage over one or more light:dark cycles.

Disruption of the homeodomain transcription factor orthopedia homeobox (Otp) is associated with obesity and anxiety.

OBJECTIVE: Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. METHODS: In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity. RESULTS: We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. OtpR108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. CONCLUSIONS: OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.

Inducible Knockout of Mouse Zfhx3 Emphasizes Its Key Role in Setting the Pace and Amplitude of the Adult Circadian Clock.

The transcription factor zinc finger homeobox 3 (ZFHX3) plays a key role in coupling intracellular transcriptional-translational oscillations with intercellular synchrony in mouse suprachiasmatic nucleus (SCN). However, like many key players in central nervous system function, ZFHX3 serves an important role in neurulation and neuronal terminal differentiation while retaining discrete additional functions in the adult SCN. Recently, using a dominant missense mutation in mouse Zfhx3, we established that this gene can modify circadian period and sleep in adult animals. Nevertheless, we were still concerned that the neurodevelopmental consequences of ZFHX3 dysfunction in this mutant may interfere with, or confound, its critical adult-specific roles in SCN circadian function. To circumvent the developmental consequences of Zfhx3 deletion, we crossed a conditional null Zfhx3 mutant to an inducible, ubiquitously expressed Cre line (B6.Cg-Tg(UBC-cre/ERT2)1Ejb/J). This enabled us to assess circadian behavior in the same adult animals both before and after Cre-mediated excision of the critical Zfhx3 exons using tamoxifen treatment. Remarkably, we found a strong and significant alteration in circadian behavior in tamoxifen-treated homozygous animals with no phenotypic changes in heterozygous or control animals. Cre-mediated excision of Zfhx3 critical exons in adult animals resulted in shortening of the period of wheel-running in constant darkness by more than 1 h in the majority of homozygotes while, in 30% of animals, excision resulted in complete behavioral arrhythmicity. In addition, we found that homozygous animals reentrain almost immediately to 6-h phase advances in the light-dark cycle. No additional overt phenotypic changes were evident in treated homozygous animals. These findings confirm a sustained and significant role for ZFHX3 in maintaining rhythmicity in the adult mammalian circadian system.