Understanding circadian dynamics: current progress and future directions for chronobiology in drug discovery.

INTRODUCTION: Most mammalian physiology is orchestrated by the circadian clock, including drug transport and metabolism. As a result, efficacy and toxicity of many drugs are influenced by the timing of their administration, which has led to the establishment of the field of chronopharmacology. AREAS COVERED: In this review, the authors provide an overview of the current knowledge about the time-of-day dependent aspects of drug metabolism and the importance of chronopharmacological strategies for drug development. They also discuss the factors influencing rhythmic drug pharmacokinetic including sex, metabolic diseases, feeding rhythms, and microbiota, that are often overlooked in the context of chronopharmacology. This article summarizes the involved molecular mechanisms and functions and explains why these parameters should be considered in the process of drug discovery. EXPERT OPINION: Although chronomodulated treatments have shown promising results, particularly for cancer, the practice is still underdeveloped due to the associated high cost and time investments. However, implementing this strategy at the preclinical stage could offer a new opportunity to translate preclinical discoveries into successful clinical treatments.

Multiomics reveals multilevel control of renal and systemic metabolism by the renal tubular circadian clock.

Circadian rhythmicity in renal function suggests rhythmic adaptations in renal metabolism. To decipher the role of the circadian clock in renal metabolism, we studied diurnal changes in renal metabolic pathways using integrated transcriptomic, proteomic, and metabolomic analysis performed on control mice and mice with an inducible deletion of the circadian clock regulator Bmal1 in the renal tubule (cKOt). With this unique resource, we demonstrated that approximately 30% of RNAs, approximately 20% of proteins, and approximately 20% of metabolites are rhythmic in the kidneys of control mice. Several key metabolic pathways, including NAD+ biosynthesis, fatty acid transport, carnitine shuttle, and ß-oxidation, displayed impairments in kidneys of cKOt mice, resulting in perturbed mitochondrial activity. Carnitine reabsorption from primary urine was one of the most affected processes with an approximately 50% reduction in plasma carnitine levels and a parallel systemic decrease in tissue carnitine content. This suggests that the circadian clock in the renal tubule controls both kidney and systemic physiology.

Disruption of the circadian clock component BMAL1 elicits an endocrine adaption impacting on insulin sensitivity and liver disease.

SignificanceWhile increasing evidence associates the disruption of circadian rhythms with pathologic conditions, including obesity, type 2 diabetes, and nonalcoholic fatty liver diseases (NAFLD), the involved mechanisms are still poorly described. Here, we show that, in both humans and mice, the pathogenesis of NAFLD is associated with the disruption of the circadian clock combined with perturbations of the growth hormone and sex hormone pathways. However, while this condition protects mice from the development of fibrosis and insulin resistance, it correlates with increased fibrosis in humans. This suggests that the perturbation of the circadian clock and its associated disruption of the growth hormone and sex hormone pathways are critical for the pathogenesis of metabolic and liver diseases.

The Mechanisms and Physiological Consequences of Diurnal Hepatic Cell Size Fluctuations: A Brief Review.

Liver size in mammals fluctuates throughout the day and correlates with changes in hepatocyte size. However, the role of these daily changes in liver and hepatocyte size and the underlying molecular mechanisms remain largely unknown. In this review, we highlight the view that hepatocyte size, and thus, overall organ size, is subject to regulation by the circadian clock and feeding/fasting cycles. To that end, we provide an overview of the current literature dealing with this phenomenon and elaborate the role of feeding and nutrients in this process. We will discuss the role of hepatic protein content and synthesis, which are both subject to diurnal regulation, in daily hepatocyte and liver size fluctuations. Although there is evidence that changes in hepatocyte and liver size are associated with daily variations in macromolecule content, there is also evidence that these changes in size may be actively regulated by modifications of the cells' osmotic environment. Future research will need to examine the intriguing possibility that hepatocyte and liver size fluctuations may be required for normal liver function and to reveal the underlying molecular mechanisms behind this process.

Systematic analysis of differential rhythmic liver gene expression mediated by the circadian clock and feeding rhythms.

The circadian clock and feeding rhythms are both important regulators of rhythmic gene expression in the liver. To further dissect the respective contributions of feeding and the clock, we analyzed differential rhythmicity of liver tissue samples across several conditions. We developed a statistical method tailored to compare rhythmic liver messenger RNA (mRNA) expression in mouse knockout models of multiple clock genes, as well as PARbZip output transcription factors (Hlf/Dbp/Tef). Mice were exposed to ad libitum or night-restricted feeding under regular light-dark cycles. During ad libitum feeding, genetic ablation of the core clock attenuated rhythmic-feeding patterns, which could be restored by the night-restricted feeding regimen. High-amplitude mRNA expression rhythms in wild-type livers were driven by the circadian clock, but rhythmic feeding also contributed to rhythmic gene expression, albeit with significantly lower amplitudes. We observed that Bmal1 and Cry1/2 knockouts differed in their residual rhythmic gene expression. Differences in mean expression levels between wild types and knockouts correlated with rhythmic gene expression in wild type. Surprisingly, in PARbZip knockout mice, the mean expression levels of PARbZip targets were more strongly impacted than their rhythms, potentially due to the rhythmic activity of the D-box-repressor NFIL3. Genes that lost rhythmicity in PARbZip knockouts were identified to be indirect targets. Our findings provide insights into the diurnal transcriptome in mouse liver as we identified the differential contributions of several core clock regulators. In addition, we gained more insights on the specific effects of the feeding-fasting cycle.

Mitochondrial gene signature in the prefrontal cortex for differential susceptibility to chronic stress.

Mitochondrial dysfunction was highlighted as a crucial vulnerability factor for the development of depression. However, systemic studies assessing stress-induced changes in mitochondria-associated genes in brain regions relevant to depression symptomatology remain scarce. Here, we performed a genome-wide transcriptomic study to examine mitochondrial gene expression in the prefrontal cortex (PFC) and nucleus accumbens (NAc) of mice exposed to multimodal chronic restraint stress. We identified mitochondria-associated gene pathways as most prominently affected in the PFC and with lesser significance in the NAc. A more detailed mitochondrial gene expression analysis revealed that in particular mitochondrial DNA-encoded subunits of the oxidative phosphorylation complexes were altered in the PFC. The comparison of our data with a reanalyzed transcriptome data set of chronic variable stress mice and major depression disorder subjects showed that the changes in mitochondrial DNA-encoded genes are a feature generalizing to other chronic stress-protocols as well and might have translational relevance. Finally, we provide evidence for changes in mitochondrial outputs in the PFC following chronic stress that are indicative of mitochondrial dysfunction. Collectively, our work reinforces the idea that changes in mitochondrial gene expression are key players in the prefrontal adaptations observed in individuals with high behavioral susceptibility and resilience to chronic stress.

MondoA regulates gene expression in cholesterol biosynthesis-associated pathways required for zebrafish epiboly.

The glucose-sensing Mondo pathway regulates expression of metabolic genes in mammals. Here, we characterized its function in the zebrafish and revealed an unexpected role of this pathway in vertebrate embryonic development. We showed that knockdown of mondoa impaired the early morphogenetic movement of epiboly in zebrafish embryos and caused microtubule defects. Expression of genes in the terpenoid backbone and sterol biosynthesis pathways upstream of pregnenolone synthesis was coordinately downregulated in these embryos, including the most downregulated gene nsdhl. Loss of Nsdhl function likewise impaired epiboly, similar to MondoA loss of function. Both epiboly and microtubule defects were partially restored by pregnenolone treatment. Maternal-zygotic mutants of mondoa showed perturbed epiboly with low penetrance and compensatory changes in the expression of terpenoid/sterol/steroid metabolism genes. Collectively, our results show a novel role for MondoA in the regulation of early vertebrate development, connecting glucose, cholesterol and steroid hormone metabolism with early embryonic cell movements.

In most animals, a protein called MondoA closely monitors the amount of glucose in the body, as this type of sugar is the fuel required for many life processes. Glucose levels also act as a proxy for the availability of other important nutrients. Once MondoA has detected glucose molecules, it turns genetic programmes on and off depending on the needs of the cell. So far, these mechanisms have mainly been studied in adult cells. However, recent studies have shown that proteins that monitor nutrient availability, and their associated pathways, can control early development. MondoA had not been studied in this context before, so Weger et al. decided to investigate its role in embryonic development. The experiments used embryos from zebrafish, a small freshwater fish whose early development is easily monitored and manipulated in the laboratory. Inhibiting production of the MondoA protein in zebrafish embryos prevented them from maturing any further, stopping their development at an early key stage. This block was caused by defects in microtubules, the tubular molecules that act like a microscopic skeleton to provide structural support for cells and guide transport of cell components. In addition, the pathway involved in the production of cholesterol and cholesterol-based hormones was far less active in embryos lacking MondoA. Treating MondoA-deficient embryos with one of these hormones corrected the microtubule defects and let the embryos progress to more advanced stages of development. These results reveal that, during development, the glucose sensor MondoA also controls pathways involved in the creation of cholesterol and associated hormones. These new insights into the metabolic regulation of development could help to understand certain human conditions; for example, certain patients with defective cholesterol pathway genes also show developmental perturbations. In addition, the work highlights a biological link between cholesterol production and cellular responses to glucose, which Weger et al. hope could one day help to identify new cholesterol-lowering drugs.

The glucocorticoid receptor in the nucleus accumbens plays a crucial role in social rank attainment in rodents.

Social hierarchy in social species is usually established through competitive encounters with conspecifics. It determines the access to limited resources and, thus, leads to reduced fights among individuals within a group. Despite the known importance of social rank for health and well-being, the knowledge about the processes underlying rank attainment remains limited. Previous studies have highlighted the nucleus accumbens (NAc) as a key brain region in the attainment of social hierarchies in rodents. In addition, glucocorticoids and the glucocorticoid receptor (GR) have been implicated in the establishment of social hierarchies and social aversion. However, whether GR in the NAc is involved in social dominance is not yet known. To address this question, we first established that expression levels of GR in the NAc of high anxious, submissive-prone rats are lower than that of their low anxious, dominant-prone counterparts. Furthermore, virally-induced downregulation of GR expression in the NAc in rats led to an improvement of social dominance rank. We found a similar result in a cell-specific mouse model lacking GR in dopaminoceptive neurons (i.e., neurons containing dopamine receptors). Indeed, when cohabitating in dyads of mixed genotypes, mice deficient for GR in dopaminoceptive neurons had a higher probability to become dominant than wild-type mice. Overall, our results highlight GR in the NAc and in dopaminoceptive neurons as an important regulator of social rank attainment.

Glucocorticoid deficiency causes transcriptional and post-transcriptional reprogramming of glutamine metabolism.

Background: Deficient glucocorticoid biosynthesis leading to adrenal insufficiency is life-threatening and is associated with significant co-morbidities. The affected pathways underlying the pathophysiology of co-morbidities due to glucocorticoid deficiency remain poorly understood and require further investigation. Methods: To explore the pathophysiological processes related to glucocorticoid deficiency, we have performed global transcriptional, post-transcriptional and metabolic profiling of a cortisol-deficient zebrafish mutant with a disrupted ferredoxin (fdx1b) system. Findings: fdx1b−/− mutants show pervasive reprogramming of metabolism, in particular of glutamine-dependent pathways such as glutathione metabolism, and exhibit changes of oxidative stress markers. The glucocorticoid-dependent post-transcriptional regulation of key enzymes involved in de novo purine synthesis was also affected in this mutant. Moreover, fdx1b−/− mutants exhibit crucial features of primary adrenal insufficiency, and mirror metabolic changes detected in primary adrenal insufficiency patients. Interpretation: Our study provides a detailed map of metabolic changes induced by glucocorticoid deficiency as a consequence of a disrupted ferredoxin system in an animal model of adrenal insufficiency. This improved pathophysiological understanding of global glucocorticoid deficiency informs on more targeted translational studies in humans suffering from conditions associated with glucocorticoid deficiency. Fund: Marie Curie Intra-European Fellowships for Career Development, HGF-programme BIFTM, Deutsche Forschungsgemeinschaft, BBSRC.

High anxiety trait: A vulnerable phenotype for stress-induced depression.

A great deal of research aims to identify risk factors related to individual vulnerability to develop stress-induced psychopathologies. Here, we summarize evidence that point at anxiety trait as a significant contributor to inter-individual differences in stress-vulnerability. Specifically, we underscore high anxiety trait as a key vulnerability phenotype. Highly anxious individuals show both behavioral alterations and cognitive deficits, along with more reactive physiological stress responses. We discuss efforts and progress towards the identification of genetic variants and polygenetic scores that explain differences in trait anxiety and vulnerability to stress. We then summarize molecular alterations in the brain of individuals with high anxiety trait that can help explaining the increased vulnerability to stress of these individuals. Variation in such systems can act as risk factors, which in combination with severe/prolonged stressful life events can pave the way towards the development of depression. Our viewpoint implies that the consideration of high anxiety trait as a key vulnerability phenotype in stress research can support the overall aim to obtain improved or novel therapeutic approaches.

Increased brain glucocorticoid actions following social defeat in rats facilitates the long-term establishment of social subordination.

Social rank is frequently established through aggressive encounters between new conspecifics. Despite increasing evidence suggesting that social rank is critical for the well-being of both humans and animals, knowledge about the factors influencing social rank remain scarce. Stress was previously shown to affect the establishment and maintenance of social hierarchies in rats. Likewise, increasing systemic corticosterone levels post-encounter in the emerging subordinate rat facilitates the long-term establishment of social subordination. Here, we investigated whether central corticosterone actions are sufficient to mediate this effect. Our data shows that, indeed, an intracerebroventricular corticosterone injection given to the emerging subordinate rat facilitates the long-term maintenance of the subordinate rank. Next, we attempted to identify a particular brain region in which enhancement of corticosterone actions could be sufficient to exert the facilitation of a long-term maintenance in the emerging subordinate brain. However, post-encounter administration of corticosterone into the basolateral amygdala, medial amygdala, lateral septum and the nucleus accumbens, brain regions selected for their implication in social rank establishment and emotional modulation of memory, did not affect long-term social subordination. Our study highlights the involvement of intracerebral corticosterone actions on the facilitation of long-lasting subordinate behavior, likely by having a modulatory role in the neurobehavioral plasticity engaged in the shaping of social subordination.

Stem cells and the circadian clock.

The circadian timing system is a complex biological network of interacting circadian clocks that regulates 24h rhythms of behavioral and physiological processes. One intriguing observation is that stem cell homeostasis is subject to circadian clock regulation. Rhythmic oscillations have been observed in a variety of embryonic and adult stem cell dependent processes, such as hematopoietic progenitor cell migration, the hair follicle cycle, bone remodeling, regenerative myogenesis and neurogenesis. This review aims to discuss the nature of the circadian clock in embryonic stem cells and how it changes during differentiation. Furthermore, it will examine how the circadian clock contributes to adult stem cell function in different tissues of the body with an emphasis on the brain and adult neurogenesis.

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

Impaired constitutive and regenerative neurogenesis in adult hyperglycemic zebrafish.

A growing body of evidence supports hyperglycemia as a putative contributor to several brain dysfunctions observed in diabetes patients, such as impaired memory capacity, neural plasticity, and neurogenic processes. Thanks to the persistence of radial glial cells acting as neural stem cells, the brain of the adult zebrafish constitutes a relevant model to investigate constitutive and injury-induced neurogenesis in adult vertebrates. However, there is limited understanding of the impact of hyperglycemia on brain dysfunction in the zebrafish model. This work aimed at exploring the impact of acute and chronic hyperglycemia on brain homeostasis and neurogenesis. Acute hyperglycemia was shown to promote gene expression of proinflammatory cytokines (il1ß, il6, il8, and tnfα) in the brain and chronic hyperglycemia to impair expression of genes involved in the establishment of the blood-brain barrier (claudin 5a, zona occludens 1a and b). Chronic hyperglycemia also decreased brain cell proliferation in most neurogenic niches throughout the forebrain and the midbrain. By using a stab wound telencephalic injury model, the impact of hyperglycemia on brain repair mechanisms was investigated. Whereas the initial step of parenchymal cell proliferation was not affected by acute hyperglycemia, later proliferation of neural progenitors was significantly decreased by chronic hyperglycemia in the injured brain of fish. Taken together, these data offer new evidence highlighting the evolutionary conserved adverse effects of hyperglycemia on neurogenesis and brain healing in zebrafish. In addition, our study reinforces the utility of zebrafish as a robust model for studying the effects of metabolic disorders on the central nervous system. J. Comp. Neurol. 525:442-458, 2017. © 2016 Wiley Periodicals, Inc.

Extensive Regulation of Diurnal Transcription and Metabolism by Glucocorticoids.

Altered daily patterns of hormone action are suspected to contribute to metabolic disease. It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism. Here, we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq, NMR spectroscopy and liquid chromatography-based methods. We observed dysregulation of metabolic pathways including glutaminolysis, the citrate and urea cycles and glyoxylate detoxification. Constant, non-rhythmic glucocorticoid treatment rescued many of these changes, with some notable exceptions among the amino acid related pathways. Surprisingly, the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns. A combination of E-box and glucocorticoid response elements is enriched in the rescued genes. This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure, revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription. Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency, even under glucocorticoid replacement therapy. Moreover, we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism.

Ferredoxin 1b (Fdx1b) Is the Essential Mitochondrial Redox Partner for Cortisol Biosynthesis in Zebrafish.

Mitochondrial cytochrome P450 (CYP) enzymes rely on electron transfer from the redox partner ferredoxin 1 (FDX1) for catalytic activity. Key steps in steroidogenesis require mitochondrial CYP enzymes and FDX1. Over 30 ferredoxin mutations have been explored in vitro; however, no spontaneously occurring mutations have been identified in humans leaving the impact of FDX1 on steroidogenesis in the whole organism largely unknown. Zebrafish are an important model to study human steroidogenesis, because they have similar steroid products and endocrine tissues. This study aimed to characterize the influence of ferredoxin on steroidogenic capacity in vivo by using zebrafish. Zebrafish have duplicate ferredoxin paralogs: fdx1 and fdx1b. Although fdx1 was observed throughout development and in most tissues, fdx1b was expressed after development of the zebrafish interrenal gland (counterpart to the mammalian adrenal gland). Additionally, fdx1b was restricted to adult steroidogenic tissues, such as the interrenal, gonads, and brain, suggesting that fdx1b was interacting with steroidogenic CYP enzymes. By using transcription activator-like effector nucleases, we generated fdx1b mutant zebrafish lines. Larvae with genetic disruption of fdx1b were morphologically inconspicuous. However, steroid hormone analysis by liquid chromatography tandem mass spectrometry revealed fdx1b mutants failed to synthesize glucocorticoids. Additionally, these mutants had an up-regulation of the hypothalamus-pituitary-interrenal axis and showed altered dark-light adaptation, suggesting impaired cortisol signaling. Antisense morpholino knockdown confirmed Fdx1b is required for de novo cortisol biosynthesis. In summary, by using zebrafish, we generated a ferredoxin knockout model system, which demonstrates for the first time the impact of mitochondrial redox regulation on glucocorticoid biosynthesis in vivo.

Glycolysis supports embryonic muscle growth by promoting myoblast fusion.

Muscles ensure locomotion behavior of invertebrate and vertebrate organisms. They are highly specialized and form using conserved developmental programs. To identify new players in muscle development we screened Drosophila and zebrafish gene expression databases for orthologous genes expressed in embryonic muscles. We selected more than 100 candidates. Among them is the glycolysis gene Pglym78/pgam2, the attenuated expression of which results in the formation of thinner muscles in Drosophila embryos. This phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, suggesting affected myoblast fusion. Indeed, a detailed analysis of developing muscles in Pglym78 RNAi embryos reveals loss of fusion-associated actin foci and an inefficient Notch decay in fusion competent myoblasts, both known to be required for fusion. In addition to Pglym78, our screen identifies six other genes involved in glycolysis or in pyruvate metabolism (Pfk, Tpi, Gapdh, Pgk, Pyk, and Impl3). They are synchronously activated in embryonic muscles and attenuation of their expression leads to similar muscle phenotypes, which are characterized by fibers with reduced size and the presence of unfused myoblasts. Our data also show that the cell size triggering insulin pathway positively regulates glycolysis in developing muscles and that blocking the insulin or target of rapamycin pathways phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. Collectively, these data suggest that setting metabolism to glycolysis-stimulated biomass production is part of a core myogenic program that operates in both invertebrate and vertebrate embryos and promotes formation of syncytial muscles.

A chemical screening procedure for glucocorticoid signaling with a zebrafish larva luciferase reporter system.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

Real-time in vivo monitoring of circadian E-box enhancer activity: a robust and sensitive zebrafish reporter line for developmental, chemical and neural biology of the circadian clock.

The circadian clock co-ordinates physiology and behavior with the day/night cycle. It consists of a transcriptional-translational feedback loop that generates self-sustained oscillations in transcriptional activity with a roughly 24h period via E-box enhancer elements. Numerous in vivo aspects of core clock feedback loop function are still incompletely understood, including its maturation during development, tissue-specific activity and perturbation in disease states. Zebrafish are promising models for biomedical research due to their high regenerative capacity and suitability for in vivo drug screens, and transgenic zebrafish lines are valuable tools to study transcriptional activity in vivo during development. To monitor the activity of the core clock feedback loop in vivo, we created a transgenic zebrafish line expressing a luciferase reporter gene under the regulation of a minimal promoter and four E-boxes. This Tg(4xE-box:Luc) line shows robust oscillating reporter gene expression both under light-dark cycles and upon release into constant darkness. Luciferase activity starts to oscillate during the first days of development, indicating that the core clock loop is already functional at an early stage. To test whether the Tg(4xE-box:Luc) line could be used in drug screens aimed at identifying compounds that target the circadian clock in vivo, we examined drug effects on circadian period. We were readily able to detect period changes as low as 0.7h upon treatment with the period-lengthening drugs lithium chloride and longdaysin in an assay set-up suitable for large-scale screens. Reporter gene mRNA expression is also detected in the adult brain and reveals differential clock activity across the brain, overlapping with endogenous clock gene expression. Notably, core clock activity is strongly correlated with brain regions where neurogenesis takes place and can be detected in several types of neural progenitors. Our results demonstrate that the Tg(4xE-box:Luc) line is an excellent tool for studying the regulation of the circadian clock and its maturation in vivo and in real time. Furthermore, it is highly suitable for in vivo screens targeting the core clock mechanism that take into account the complexity of an intact organism. Finally, it allows mapping of clock activity in the brain of a vertebrate model organism with prominent adult neurogenesis and high regeneration capacity.

The circadian clock and glucocorticoids--interactions across many time scales.

Glucocorticoids are steroid hormones of the adrenal gland that are an integral component of the stress response and regulate many physiological processes, including metabolism and immune response. Their release into the blood is highly dynamic and occurs in about hourly pulses, the amplitude of which is modulated in a daytime dependent fashion. In addition, in many species seasonal changes in basal glucocorticoid levels have been reported. In their target tissues, glucocorticoids bind to cytoplasmic receptors of the nuclear receptor superfamily. Upon binding, these receptors regulate transcription in a highly dynamic fashion, which involves stochastic binding to regulatory DNA elements on a time scale of seconds and heat shock protein mediated receptor-ligand complex recycling within minutes. The glucocorticoid hormone system interacts with another highly dynamic system, the circadian clock. The circadian clock is an endogenous biological timing mechanism that allows organisms to anticipate regular daily changes in their environment. It regulates daily rhythms of glucocorticoid release by a variety of mechanisms, modulates glucocorticoid signaling and is itself influenced by glucocorticoids. Here, we discuss mechanisms, functions and interactions of the circadian and glucocorticoid systems across time scales ranging from seconds (DNA binding by transcriptional regulators) to years (seasonal rhythms).