Functional central rhythmicity and light entrainment, but not liver and muscle rhythmicity, are Clock independent

The circadian rhythmicity of hormone secretion, body temperature, and sleep/wakefulness results from an endogenous rhythm of neural activity generated by clock genes in the suprachiasmatic nucleus (SCN). One of these genes, Clock, has been considered essential for the generation of cellular rhythmicity centrally and in the periphery; however, melatonin-proficient Clock(Delta19) + MEL mutant mice retain melatonin rhythmicity, suggesting that their central rhythmicity is intact. Here we show that melatonin production in these mutants was rhythmic in constant darkness and could be entrained by brief single daily light pulses. Under normal light-dark conditions, per2 and prokineticin2 (PK2) mRNA expression was rhythmic in the SCN of Clock(Delta19) + MEL mice. Expression of Bmal1 and npas2 was not altered, whereas per1 expression was arrhythmic. In contrast to the SCN, per1 and per2 expression, as well as Bmal1 expression in liver and skeletal muscle, together with plasma corticosterone, was arrhythmic in Clock(Delta19) + MEL mutant mice in normal light-dark conditions. npas2 mRNA was also arrhythmic in liver but rhythmic in muscle. The Clock(Delta19) mutation does not abolish central rhythmicity and light entrainment, suggesting that a functional Clock homolog, possibly npas2, exists in the SCN. Nevertheless, the SCN of Clock(Delta19) + MEL mutant mice cannot maintain liver and muscle rhythmicity through rhythmic outputs, including melatonin secretion, in the absence of functional Clock expression in the tissues. Therefore, liver and muscle, but not SCN, have an absolute requirement for CLOCK, with as yet unknown Clock-independent factors able to generate the latter.     

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.     

Cell Type-Specific Functions of Period Genes Revealed by Novel Adipocyte and Hepatocyte Circadian Clock Models

In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.     

Clock gene daily profiles and their phase relationship in the rat suprachiasmatic nucleus are affected by photoperiod

Rhythmicity of the rat suprachiasmatic nucleus (SCN), a site of the circadian pacemaker, is affected by daylength; that is, by the photoperiod. Whereas various markers of rhythmicity have been followed, so far there have been no studies on the effect of the photoperiod on the expression of the clock genes in the rat SCN. To fill the gap and to better understand the photoperiodic modulation of the SCN state, rats were maintained either under a long photoperiod with 16 h of light and 8 h of darkness per day (LD16:8) or under a short LD8:16 photoperiod, and daily profiles of Per1, Cry1, Bmal1 and Clock mRNA in darkness were assessed by in situ hybridization method. The photoperiod affected phase, waveform, and amplitude of the rhythmic gene expression as well as phase relationship between their profiles. Under the long period, the interval of elevated Per1 mRNA lasted for a longer and that of elevated Bmal1 mRNA for a shorter time than under the short photoperiod. Under both photoperiods, the morning and the daytime Per1 and Cry1 mRNA rise as well as the morning Bmal1 mRNA decline were closely linked to the morning light onset. Amplitude of Per1, Cry1, and Bmal1 mRNA rhythms was larger under the short than under the long photoperiod. Also, under the short photoperiod, the daily Clock mRNA profile exhibited a significant rhythm. Altogether, the data indicate that the whole complex molecular clockwork in the rat SCN is photoperiod dependent and hence may differ according to the season of the year.     

Photic stimulation of the suprachiasmatic nucleus via the non-visual optic system. A gene expression study in the blind Crx −/− mouse

The visual system of vertebrates consists of an image-forming and a non-image-forming optic system; the image-forming optic system involves the classic photoreceptors, the rods and cones, whereas the non-image-forming optic system involves the melanopsin-containing retinal ganglion cells. Both optic systems make direct neuroanatomical connections to the suprachiasmatic nucleus (SCN) in the hypothalamus in which the biological clock of vertebrates is located. The rhythmic output from SCN neurons is entrained by light via the retina and the retinohypothalamic tract. The response of exposure to light during the subjective night is an immediate expression of several early response genes in the SCN. We show, by quantitative real-time polymerase chain reaction, that the amount of melanopsin mRNA in the retinal ganglion cells is preserved in the blind Crx ^?/? mouse with degenerated classic photoreceptors. At zeitgeber time 16, the Crx ^?/? and wild-type mice were exposed to 1 h of light. This resulted in a strong up-regulation of the immediate early genes Nr4a1, Erg, and Rrad in the SCN of both genotypes. Light stimulation during the subjective night resulted in a strong up-regulation of c-fos in both genotypes with a significantly higher up-regulation in the blind Crx ^?/? mouse. Expression of Grp and Vip, the genes for two classic peptides located in the SCN, was not influenced by light stimulation. The data strongly indicate the involvement of the melanopsin-based non-visual optic system in the regulation of immediate early genes in the SCN.     

Melatonin Signaling Modulates Clock Genes Expression in the Mouse Retina

Previous studies have shown that retinal melatonin plays an important role in the regulation of retinal daily and circadian rhythms. Melatonin exerts its influence by binding to G-protein coupled receptors named melatonin receptor type 1 and type 2 and both receptors are present in the mouse retina. Earlier studies have shown that clock genes are rhythmically expressed in the mouse retina and melatonin signaling may be implicated in the modulation of clock gene expression in this tissue. In this study we determined the daily and circadian expression patterns of Per1, Per2, Bmal1, Dbp, Nampt and c-fos in the retina and in the photoreceptor layer (using laser capture microdissection) in C3H-f+/+ and in melatonin receptors of knockout (MT₁ and MT₂) of the same genetic background using real-time quantitative RT-PCR. Our data indicated that clock and clock-controlled genes are rhythmically expressed in the retina and in the photoreceptor layer. Removal of melatonin signaling significantly affected the pattern of expression in the retina whereas in the photoreceptor layer only the Bmal1 circadian pattern of expression was affected by melatonin signaling removal. In conclusion, our data further support the notion that melatonin signaling may be important for the regulation of clock gene expression in the inner or ganglion cells layer, but not in photoreceptors.     

Circadian Oscillations of Molecular Clock Components in the Cerebellar Cortex of the Rat

The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum. (Author correspondence: mrath@sund.ku.dk)     

Clock Genes Influence Gene Expression in Growth Plate and Endochondral Ossification in Mice

We have previously shown transient promotion by parathyroid hormone of Period-1 (Per1) expression in cultured chondrocytes. Here we show the modulation by clock genes of chondrogenic differentiation through gene transactivation of the master regulator of chondrogenesis Indian hedgehog (IHH) in chondrocytes of the growth plate. Several clock genes were expressed with oscillatory rhythmicity in cultured chondrocytes and rib growth plate in mice, whereas chondrogenesis was markedly inhibited in stable transfectants of Per1 in chondrocytic ATDC5 cells and in rib growth plate chondrocytes from mice deficient of brain and muscle aryl hydrocarbon receptor nuclear translocator-like (BMAL1). Ihh promoter activity was regulated by different clock gene products, with clear circadian rhythmicity in expression profiles of Ihh in the growth plate. In BMAL1-null mice, a predominant decrease was seen in Ihh expression in the growth plate with a smaller body size than in wild-type mice. BMAL1 deficit led to disruption of the rhythmic expression profiles of both Per1 and Ihh in the growth plate. A clear rhythmicity was seen with Ihh expression in ATDC5 cells exposed to dexamethasone. In young mice defective of BMAL1 exclusively in chondrocytes, similar abnormalities were found in bone growth and Ihh expression. These results suggest that endochondral ossification is under the regulation of particular clock gene products expressed in chondrocytes during postnatal skeletogenesis through a mechanism relevant to the rhythmic Ihh expression.     

Circadian oscillators in the mouse brain: molecular clock components in the neocortex and cerebellar cortex

The circadian timekeeper of the mammalian brain resides in the suprachiasmatic nucleus of the hypothalamus (SCN), and is characterized by rhythmic expression of a set of clock genes with specific 24-h daily profiles. An increasing amount of data suggests that additional circadian oscillators residing outside the SCN have the capacity to generate peripheral circadian rhythms. We have recently shown the presence of SCN-controlled oscillators in the neocortex and cerebellum of the rat. The function of these peripheral brain clocks is unknown, and elucidating this could involve mice with conditional cell-specific clock gene deletions. This prompted us to analyze the molecular clockwork of the mouse neocortex and cerebellum in detail. Here, by use of in situ hybridization and quantitative RT-PCR, we show that clock genes are expressed in all six layers of the neocortex and the Purkinje and granular cell layers of the cerebellar cortex of the mouse brain. Among these, Per1, Per2, Cry1, Arntl, and Nr1d1 exhibit circadian rhythms suggesting that local running circadian oscillators reside within neurons of the mouse neocortex and cerebellar cortex. The temporal expression profiles of clock genes are similar in the neocortex and cerebellum, but they are delayed by 5 h as compared to the SCN, suggestively reflecting a master–slave relationship between the SCN and extra-hypothalamic oscillators. Furthermore, ARNTL protein products are detectable in neurons of the mouse neocortex and cerebellum, as revealed by immunohistochemistry. These findings give reason to further pursue the physiological significance of circadian oscillators in the mouse neocortex and cerebellum.     

Melatonin administered during the fetal stage affects circadian clock in the suprachiasmatic nucleus but not in the liver

The mammalian circadian system develops gradually during ontogenesis, and after birth, the system is already set to a phase of the mothers. The role of maternal melatonin in the entrainment of fetal circadian clocks has been suggested, but direct evidence is lacking. In our study, intact or pinealectomized pregnant rats were exposed to constant light (LL) throughout pregnancy to suppress the endogenous melatonin and behavioral rhythms. During the last 5 days of gestation, the rats were injected with melatonin or vehicle or were left untreated. After delivery, daily expression profiles of c‐fos and Avp in the suprachiasmatic nuclei (SCN), and Per1, Per2, Rev‐erbα, and Bmal1 in the liver were measured in 1‐day‐old pups. Due to the LL exposure, no gene expression rhythms were detected in the SCN of untreated pregnant rats or in the SCN and liver of the pups. The administration of melatonin to pregnant rats entrained the pups' gene expression profiles in the SCN, but not in the liver. Melatonin did not affect the maternal behavior during pregnancy. Vehicle injections also synchronized the gene expression in the SCN but not in the liver. Melatonin and vehicle entrained the gene expression profiles to different phases, demonstrating that the effect of melatonin was apparently not due to the treatment procedure per se. The data demonstrate that in pregnant rats with suppressed endogenous melatonin levels, pharmacological doses of melatonin affect the fetal clock in the SCN but not in the liver. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 131–144, 2015