Clock genes in cell clocks: roles, actions, and mysteries

Cellular events must be organized in the time dimension as well as in the space dimension for many proteins to perform their cellular functions effectively. The intracellular molecular oscillating loops that compose the cell's circadian clock coordinate the timing of the expression of a variety of genes with basic or specific cellular functions. In mammals, the temporal pattern of clock gene expression generated in each SCN neuron is coupled to those of other cells and, amplified, spreads its signals through the brain and then, via feeding behavior, glucocorticoids, and sympathetic nerves, to peripheral organs. These peripheral organs have their own circadian clocks. In some tissues, such as liver, there is also a clock-regulating cell cycle, which interacts strongly with the components and temporal organization of the circadian clock. Some tissues, however, such as testis, express clock genes whose function, if any, remains unclear. Furthermore, circadian clock function may be suspended in differentiating tissue. Thus, the prominence of circadian organization may not apply equally to all tissues under all conditions.     

Circadian expression of clock genes in human peripheral leukocytes

In mammals, behavioral and physiological processes display 24-h rhythms that are regulated by a circadian system. In the present study, we investigated the possibility that the expression of clock genes in peripheral leukocytes can be used to assess the circadian clock system. We found that Per1 and Per2 exhibit circadian oscillations in mRNA expression in mouse peripheral leukocytes. Furthermore, the rhythms of Per1 and Per2 mRNA expression in peripheral leukocytes are severely blunted in homozygous Cry1/2 double-deficient mice that are known to have an abolished biological clock. We have examined the circadian expression of clock genes in human leukocytes and found that Per1 mRNA exhibits a robust circadian expression while Per2 and Bmal1 mRNA showed weak rhythm. These observations suggest that monitoring Per1 mRNA expression in human leukocytes may be useful for investigating the function of the circadian system in physiological and pathophysiological states.     

Transplanted Drosophila excretory tubules maintain circadian clock cycling out of phase with the host

Circadian rhythms in behaviors and physiological processes are driven by conserved molecular mechanisms involving the rhythmic expression of clock genes in the brains of animals [1]. The persistence of similar molecular rhythms in peripheral tissues in vitro [2] [3] suggests that these tissues contain self-sustained circadian clocks that may be linked to rhythmic physiological functions. It is not known how brain and peripheral clocks are organized into a synchronized timing system; however, it has been assumed that peripheral clocks submit to a master clock in the brain. To address this matter we examined the expression of two clock genes, period (per) and timeless (tim), in host and transplanted abdominal organs of Drosophila. We found that excretory organs in tissue culture display free-running, light-sensitive oscillations in per and tim gene activity indicating that they house self-sustained circadian clocks. To test for humoral factors, we monitored cycling of the TIM protein in excretory tubules transplanted into host flies entrained to an opposite light-dark cycle. We show that the clock protein in the donor tubules cycled out of phase with that in the host tubules, indicating that different organs may cycle independently, despite sharing the same hormonal milieu. We suggest that one way to achieve circadian coordination of physiological sub-systems in higher animals may be through the direct entrainment of light-sensitive clocks by environmental signals.     

Feeding and circadian clocks

The mammalian genome encodes at least a dozen of genes directly involved in the regulation of the feedback loops constituting the circadian clock. The circadian system is built up on a multitude of oscillators organized according to a hierarchical model in which neurons of the suprachiasmatic nuclei of the hypothalamus may drive the central circadian clock and all the other somatic cells may possess the molecular components allowing tissues and organs to constitute peripheral clocks. Suprachiasmatic neurons are driving the central circadian clock which is reset by lighting cues captured and integrated by the melanopsin cells of the retina and define the daily rhythms of locomotor activity and associated physiological regulatory pathways like feeding and metabolism. This central clock entrains peripheral clocks which can be synchronized by non-photic environmental cues and uncoupled from the central one depending on the nature and the strength of the circadian signal. The human circadian clock and its functioning in central or peripheral tissues are currently being explored to increase the therapeutic efficacy of timed administration of drugs or radiation, and to offer better advice on lighting and meal timing useful for frequent travelers suffering from jet lag and for night workers' comfort. However, the molecular mechanism driving and coordinating the central and peripheral clocks through a wide range of synchronizers (lighting, feeding, physical or social activities) remains a mystery.     

Timed maternal melatonin treatment reverses circadian disruption of the fetal adrenal clock imposed by exposure to constant light

Surprisingly, in our modern 24/7 society, there is scant information on the impact of developmental chronodisruption like the one experienced by shift worker pregnant women on fetal and postnatal physiology. There are important differences between the maternal and fetal circadian systems; for instance, the suprachiasmatic nucleus is the master clock in the mother but not in the fetus. Despite this, several tissues/organs display circadian oscillations in the fetus. Our hypothesis is that the maternal plasma melatonin rhythm drives the fetal circadian system, which in turn relies this information to other fetal tissues through corticosterone rhythmic signaling. The present data show that suppression of the maternal plasma melatonin circadian rhythm, secondary to exposure of pregnant rats to constant light along the second half of gestation, had several effects on fetal development. First, it induced intrauterine growth retardation. Second, in the fetal adrenal in vivo it markedly affected the mRNA expression level of clock genes and clock-controlled genes as well as it lowered the content and precluded the rhythm of corticosterone. Third, an altered in vitro fetal adrenal response to ACTH of both, corticosterone production and relative expression of clock genes and steroidogenic genes was observed. All these changes were reversed when the mother received a daily dose of melatonin during the subjective night; supporting a role of melatonin on overall fetal development and pointing to it as a 'time giver' for the fetal adrenal gland. Thus, the present results collectively support that the maternal circadian rhythm of melatonin is a key signal for the generation and/or synchronization of the circadian rhythms in the fetal adrenal gland. In turn, low levels and lack of a circadian rhythm of fetal corticosterone may be responsible of fetal growth restriction; potentially inducing long term effects in the offspring, possibility that warrants further research.     

Gene duplication and complex circadian clocks in mammals

The circadian clock arose early in the evolution of life to enable organisms to adapt to the cycle of day and night. Recently, the extent and importance of circadian regulation of behaviour and physiology has come to be more fully realized. Core molecular cogs of circadian oscillators appear to have been largely conserved between such diverse organisms as Drosophila melanogaster and mammals. However, gene duplication events have produced multiple copies of many clock genes in mammals. Recent studies suggest that genome duplication has lead to increased circadian complexity and local tissue regulation. This has important implications for temporal regulation of behaviour via multiple clocks in the central nervous system, and also extends to the local physiology of major body organs and tissues.     

A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands

In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co-culture system is a useful tool to analyze humoral communication between different tissues or cell populations.     

Association between circadian clock genes and diapause incidence in Drosophila triauraria

Diapause is an adaptive response triggered by seasonal photoperiodicity to overcome unfavorable seasons. The photoperiodic clock is a system that controls seasonal physiological processes, but our knowledge about its physiological mechanisms and genetic architecture remains incomplete. The circadian clock is another system that controls daily rhythmic physiological phenomena. It has been argued that there is a connection between the two clocks. To examine the genetic connection between them, we analyzed the associations of five circadian clock genes (period, timeless, Clock, cycle and cryptochrome) with the occurrence of diapause in Drosophila triauraria, which shows a robust reproductive diapause with clear photoperiodicity. Non-diapause strains found in low latitudes were compared in genetic crosses with the diapause strain, in which the diapause trait is clearly dominant. Single nucleotide polymorphism and deletion analyses of the five circadian clock genes in backcross progeny revealed that allelic differences in timeless and cryptochrome between the strains were additively associated with the differences in the incidence of diapause. This suggests that there is a molecular link between certain circadian clock genes and the occurrence of diapause.     

Roles of circadian clock genes in insect photoperiodism

Functional involvement of a circadian clock in photoperiodism for measuring the length of day or night had been proposed more than 70 years ago, and various physiological experiments have supported the idea. However, the molecular basis of a circadian clock has remained veiled in insects. Nevertheless, our knowledge of the functional elements of a circadian clock governing circadian rhythmicity has advanced rapidly. Since both circadian rhythms and photoperiodism depend on the daily cycles of environmental changes, it is easy to assume that the same clock elements are involved in both processes. Recently, the RNA interference (RNAi) technique clarified that the molecular machinery of a circadian clock governing photoperiodism is identical to that governing circadian rhythmicity. Here, I review the theoretical background of photoperiodic responses incorporating a circadian clock(s) and recent progress on the molecular clockwork involved in photoperiodism in the bean bug Riptortus pedestris and other insect species. I have focused on the intense controversy regarding the involvement of a circadian clock in insect photoperiodism.     

Opposing effects of circadian clock genes bmal1 and period2 in regulation of VEGF-dependent angiogenesis in developing zebrafish

Molecular mechanisms underlying circadian-regulated physiological processes remain largely unknown. Here, we show that disruption of the circadian clock by both constant exposure to light and genetic manipulation of key genes in zebrafish led to impaired developmental angiogenesis. A bmal1-specific morpholino inhibited developmental angiogenesis in zebrafish embryos without causing obvious nonvascular phenotypes. Conversely, a period2 morpholino accelerated angiogenic vessel growth, suggesting that Bmal1 and Period2 display opposing angiogenic effects. Using a promoter-reporter system consisting of various deleted vegf-promoter mutants, we show that Bmal1 directly binds to and activates the vegf promoter via E-boxes. Additionally, we provide evidence that knockdown of Bmal1 leads to impaired Notch-inhibition-induced vascular sprouting. These results shed mechanistic insight on the role of the circadian clock in regulation of developmental angiogenesis, and our findings may be reasonably extended to other types of physiological or pathological angiogenesis.     

Insect circadian clocks: is it all in their heads?

Circadian rhythms are ubiquitous in living organisms, synchronizing life functions at the biochemical, physiological, and behavioral levels. The rhythm-generating mechanisms, collectively known as circadian clocks, are not fully understood in any organism. Research in the fruit fly Drosophila has led to the identification of several clock genes that are involved in the function of the brain-centered clock, which controls behavioral rhythms of adult flies. With the use of clock genes as markers, putative circadian clocks were mapped in the fly peripheral organs and shown to be independent from clocks located in the brain. A homologue of fruit fly period gene has been identified in moths and other insects, allowing investigations of this gene's role in known insect rhythms. This approach may increase our understanding of how circadian clocks are organized into the circadian system that orchestrates temporal integration of life processess in insects.     

The circadian clock of cyanobacteria

A circadian clock, with physiological characteristics similar to those of eukaryotes, functions in the photosynthetic prokaryote, cyanobacteria. The molecular mechanism of this clock has been efficiently dissected using a luciferase reporter gene that reports the status of the clock. A circadian clock gene cluster, kaiABC, has been cloned via rhythm mutants of cyanobacterium, Synechococcus, and many clock mutations mapped to the three kai genes. Although kai genes do not share any homology with clock genes so far identified in eukaryotes, analysis of their expression suggests that a negative feedback control of kaiC expression by KaiC generates the circadian oscillation and that KaiA functions as a positive factor to sustain this oscillation. BioEssays 22:10-15, 2000.     

Circadian physiological rhythms

Mammals show numerous circadian rhythms, some exogenous but nonetheless useful, some endogenous. Even in the absence of exogenous synchronizers, different physiological functions usually preserve their normal temporal relations, suggesting that a single clock controls most or all of the rhythmic manifestations. The role of adrenal corticosteroids and of temperature as means whereby the clock influences other functions is considered.