Verdun M. King

Circadian Cycling of the Mouse Liver Transcriptome, as Revealed by cDNA Microarray, Is Driven by the Suprachiasmatic Nucleus

BACKGROUND Genes encoding the circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) of mammals have recently been identified, but the molecular basis of circadian timing in peripheral tissue is not well understood. We used a custom-made cDNA microarray to identify mouse liver transcripts that show circadian cycles of abundance under constant conditions. RESULTS Using two independent tissue sampling and hybridization regimes, we show that approximately 9% of the 2122 genes studied show robust circadian cycling in the liver. These transcripts were categorized by their phase of abundance, defining clusters of day- and night-related genes, and also by the function of their products. Circadian regulation of genes was tissue specific, insofar as novel rhythmic liver genes were not necessarily rhythmic in the brain, even when expressed in the SCN. The rhythmic transcriptome in the periphery is, nevertheless, dependent on the SCN because surgical ablation of the SCN severely dampened or destroyed completely the cyclical expression of both canonical circadian genes and novel genes identified by microarray analysis. CONCLUSIONS Temporally complex, circadian programming of the transcriptome in a peripheral organ is imposed across a wide range of core cellular functions and is dependent on an interaction between intrinsic, tissue-specific factors and extrinsic regulation by the SCN central pacemaker.

Photoperiodic Control of Seasonality in Birds

This review examines how birds use the annual cycle in photoperiod to ensure that seasonal events—breeding, molt, and song production—happen at the appropriate time of year. Differences in breeding strategies between birds and mammals reflect basic differences in biology. Avian breeding seasons tend to be of shorter duration and more asymmetric with respect to changes in photoperiod. Breeding seasons can occur at the same time each year (predictable) or at different times (opportunistic), depending on the food resource. In all cases, there is evidence for involvement of photoperiodic control, nonphotoperiodic control, and endogenous circannual rhythmicity. In predictable breeders (most nontropical species), photoperiod is the predominant proximate factor. Increasing photoperiods of spring stimulate secretion of gonadotropin-releasing hormone (GnRH) and consequent gonadal maturation. However, breeding ends before the return of short photoperiods. This is the consequence of a second effect of long photoperiods—the induction of photorefractoriness. This dual role of long photoperiods is required to impart the asymmetry in breeding seasons. Typically, gonadal regression through photorefractoriness is associated with a massive decrease in hypothalamic GnRH, essentially a reversal to a pre-pubertal condition. Although breeding seasons are primarily determined by photoperiodic control of GnRH neurons, prolactin may be important in determining the exact timing of gonadal regression. In tropical and opportunistic breeders, endogenous circannual rhythmicity may be more important. In such species, the reproductive system remains in a state of “readiness to breed” for a large part of the year, with nonphotic cues acting as proximate cues to time breeding. Circannual rhythmicity may result from a temporal sequence of different physiological states rather than a molecular or cellular mechanism as in circadian rhythmicity. Avian homologues of mammalian clock genes Per2, Per3, Clock, bmal1, and MOP4 have been cloned. At the molecular level, avian circadian clocks appear to function in a similar manner to those of mammals. Photoperiodic time measurement involves interaction between a circadian rhythm of photoinducibility and, unlike mammals, deep brain photoreceptors. The exact location of these remains unclear. Although the eyes and pineal generate a daily cycle in melatonin, this photoperiodic signal is not used to time seasonal breeding. Instead, photoperiodic responses appear to involve direct interaction between photoreceptors and GnRH neurons. Thyroid hormones are required in some way for this system to function. In addition to gonadal function, song production is also affected by photoperiod. Several of the nuclei involved in the song system show seasonal changes in volume, greater in spring than in the fall. The increase in volume is, in part, due to an increase in cell number as a result of neurogenesis. There is no seasonal change in the birth of neurons but rather in their survival. Testosterone and melatonin appear to work antagonistically in regulating volume.

Effects of Chronic Jet Lag on Tumor Progression in Mice

Frequent transmeridian flights or predominant work at night can increase cancer risk. Altered circadian rhythms also predict for poor survival in cancer patients, whereas physical destruction of the suprachiasmatic nuclei (SCN), the hypothalamic circadian pacemaker, accelerates tumor growth in mice. Here we tested the effect of functional disruption of circadian system on tumor progression in a novel experimental model of chronic jet lag. B6D2F1 mice were synchronized with 12 hours of light and 12 hours of darkness or underwent repeat 8-hour advances of the light/dark cycle every 2 days before inoculation of Glasgow osteosarcoma. The 24-hour changes were assessed for plasma corticosterone, clock protein mPER1 expression in the SCN, and mRNA expression of clock genes mPer2 and mRev-erbα in liver and tumor. Time series were analyzed by spectral analysis and/or Cosinor. Differences were compared with analysis of variance (ANOVA). The 24-hour rest/activity cycle was ablated, and the rhythms of body temperature, serum corticosterone, and mPER1 protein expression in the SCN were markedly altered in jet-lagged mice as compared with controls (ANOVA, P < 0.001 for corticosterone and P = 0.01 for mPER1). Tumor grew faster in the jet-lagged animals as compared with controls (ANOVA, P < 0.001), whereas exposure to constant light or darkness had no effect (ANOVA, P = 0.66 and P = 0.8, respectively). The expression of mPer2 and mRev-erbα mRNAs in controls showed significant circadian rhythms in the liver (P = 0.006 and P = 0.003, respectively, Cosinor) and in the tumor (P = 0.04 and P < 0.001). Both rhythms were suppressed in the liver (P = 0.2 and P = 0.1, respectively, Cosinor) and in the tumor (P = 0.5) of jet-lagged mice. Altered environmental conditions can disrupt circadian clock molecular coordination in peripheral organs including tumors and play a significant role in malignant progression.

Glucocorticoid signaling synchronizes the liver circadian transcriptome.

Circadian control of physiology is mediated by local, tissue‐based clocks, synchronized to each other and to solar time by signals from the suprachiasmatic nuclei (SCN), the master oscillator in the hypothalamus. These local clocks coordinate the transcription of key pathways to establish tissue‐specific daily metabolic programs. How local transcriptomes are synchronized across the organism and their relative contribution to circadian output remain unclear. In the present study we showed that glucocorticoids alone are able to synchronize expression of about 60% of the circadian transcriptome. We propose that synchronization occurs directly by the action of glucocorticoids on a diverse range of downstream targets and indirectly by regulating the core clock genes mPer1, Bmal1, mCry1, and Dbp. We have identified the pivotal liver transcription factor, HNF4α, as a mediator of circadian and glucocorticoid‐regulated transcription, showing that it is a key conduit for downstream targeting. Conclusion: We have demonstrated that by orchestrating transcriptional cascades, glucocorticoids are able to direct synchronization of a diverse range of functionally important circadian genes. (HEPATOLOGY 2007;45:1478–1488.)

Disruption of circadian coordination accelerates malignant growth in mice.

An animal model (mice B6D2F1) was developed to study the consequence of suprachiasmatic nuclei (SCN) destruction on tumor growth. SCN destruction abolished the rest-activity and body temperature rhythms and markedly altered the rhythms in serum corticosterone concentration and lymphocyte count. Tumor growth was faster in mice with lesioned SCN than in controls for both tumor models studied, Glasgow osteosarcoma (GOS) and pancreatic adenocarcinoma (P03). This shows that disruption of circadian coordination accelerates malignant growth in mice, suggesting that the host circadian clock controls tumor progression.

Melatonin and Photoperiodic Time Measurement in Japanese Quail (Coturnix coturnix japonica)

Artificial extension of the duration of nocturnally secreted circulating melatonin with exogenous injections produces a short day effect in the reproductive status of mammals, and this paradigm has been applied to Japanese quail to test the hypothesis that birds are similar to mammals in this respect. Male quail reared on non-stimulatory short days (8L: 16D) were switched to mildly stimulatory 12L: 12D and given daily melatonin injections at dusk (10 μg 2 h before dusk and 10 μg at dusk) or at dawn (10 ug 2 h before dawn and 10 μm at dawn) for about 3 weeks. Although assay of circulating melatonin suggested that injections had extended the melatonin signal, there was no short day effect, i.e. reproductive stimulation was not prevented. This reinforces the view that, unlike mammals, birds do not read the duration of the melatonin signal to measure scotoperiod. Paradoxically, however, the injections resulted in a small but significant stimulation. The results are discussed in view of the postulated role for melatonin as an internal Zeitgeber, which is coupled to the external photic Zeitgeber, to regulate the circadian system.

Copulation activates Fos-like immunoreactivity in the male quail forebrain

It has been demonstrated using Fos immunocytochemistry that copulation activates specific cell populations in the mammalian brain. Prior to this study, no similar work has been carried out in birds. In mammals, Fos has identified brain circuits activated by genital (penile)/somatosensory and by olfactory/vomeronasal stimuli. Such inputs, of course, should play little or no role in birds (no penis, little or no role for olfaction) and a differential responsiveness could therefore be expected. Male Japanese quail (Coturnix japonica) were allowed to interact freely with adult females and the presence of active sexual behavior, including cloacal contact movements, was confirmed in each case. Control subjects were exposed to a domestic chick (same size as an adult quail) and no sexual behavior was observed. Copulation induced the appearance of Fos-like immunoreactive (FLI) cells in the preoptic area, the hyperstriatum ventrale, parts of the archistriatum, and the nucleus intercollicularis. Induction of FLI cells was observed throughout the rostral to caudal extent of the preoptic region of males from the level of the tractus septomesencephalicus to the level of the anterior commissure, and in the rostral part of the hypothalamus to the level of the supraoptic decussation. The FLI cells did not lie directly adjacent to the third ventricle, but were located 500-1000 microns from the ventricle wall at the level of the lateral edge of the medial preoptic nucleus or, in more caudal sections, in a position ventrolateral to the bed nucleus striae terminalis. It is unlikely that the Fos induction in males resulted from copulation-induced endocrine changes because copulation did not affect plasma levels of luteinizing hormone or testosterone. It is concluded that the responses were due to copulation-associated somatosensory inputs and/or to stimuli originating from the female.

A Direct Comparison of Photoperiodic Time Measurement and the Circadian System in European Starlings and Japanese Quail

The extent to which circadian rhythms are involved in photoperiodic time measurement in quail is enigmatic, and earlier investigations have produced results consistent with an hourglass clock or one involving damped circadian oscillators. To address the problem further, the present authors carried out a direct comparison between the clocks in quail and those in starlings. Starlings possess strongly self-sustaining circadian oscillators. In Experiment 1, comparisons of testicular growth were made between the two species when birds were exposed to light:dark (LD) 6:30, LD 6:18, and LD 18:6. Starlings grew their testes rapidly under both LD 6:30 and LD 18:6, and they became photorefractory (under LD 6:18, the testes remained undeveloped). Quail grew their testes rapidly under LD 18:6 but did not do so under LD 6:30 or LD 6:18. In Experiment 2, entrainment of the activity rhythm under cycles of LD 6:30 was investigated by measuring the phase of the rhythm after release into constant darkness (DD). Birds were exposed to either 10 cycles or 11 cycles of LD 6:30 prior to DD. Starlings maintained their 24-h rhythmicity under LD 6:30 and always free ran from the phase of the subjective day By contrast, quail showed circadian activity approximately 24 h after every light pulse and free ran from the phase of the last light pulse received. In Experiment 3, phase response curves (PRCs) were generated to 6-h light pulses. The species were strikingly different; starlings produced a Type 1 PRC, whereas quail produced a Type 0 PRC. More important, in quail the 6-h light pulse had the same effect regardless of circadian time and in almost every case activity free ran from the position of the 6-h light pulse. The results in quail are consistent with the photoperiodic time measurement system being based on a weakly self-sustaining (rapidly damping) circadian system that is invariably reset by 6 h of light, whereas in starlings the pacemakers are strongly self-sustaining. The results support the notion that hourglass pacemakers can be highly damped circadian pacemakers.

Does Unusual Entrainment of the Circadian System Under T36h Photocycles Reduce the Critical Daylength for Photoperiodic Induction in Japanese Quail

In photoperiodic species, short daylength resonance cycles of modulo t + ½t (t = 24 h) behave like long days because they entrain the circadian system so that alternate light pulses coincide with the photoinducible phase (ø1) in castrated quail. However, while a long-day response after exposure to a single long daylength is readily detected by a rise in plasma LH (photoinduction), long-term exposure to LD 6:30 is ineffective in this respect. To discover whether this occurs because of unusual entrainment, circadian rhythms in quail and starlings were investigated. Whereas starlings entrained in the expected way with alternate pulses falling at different circadian phases, activity bouts in quail appeared to follow 24 h after successive light pulses. Because of this, activity was examined in free-running conditions to confirm that the pacemaker in quail was indeed being reset to a constant phase (reset to circadian time [CT] 0) by successive pulses. Examination of the circadian rhythms of plasma melatonin secretion under LD 6:30 also showed a resetting to CT 0. The positioning of all light pulses at the same circadian phase in the early subjective day explains the lack of photoinduction in quail since ø1 in the early subjective night phase remains unilluminated. A second feature in quail is that when the length of the photophase is gradually increased within T36h cycles, there is a progressive increase in the degree of photoinduction although the photophase length remains well below the critical daylength for induction in normal T24h cycles. We therefore tested whether ø1 is reset to a constant phase by successive pulses in LD 6:30, and that this phase is also advanced relative to light onset so that photophases shorter than the critical daylength can interact with ø1 to cause induction. Such a reduction in critical daylength relative to successive LD 6:30 pulses was confirmed by transferring quail to various types of long day and measuring the change in LH secretion. When the long-day test was replaced with continuous light, stimulation of LH secretion occurred 5-7 h earlier in quail pretreated with LD 6:30 and LD 6:54 compared to quail pretreated with LD 6:18 or LD 6:42, implying that ø 1 had been markedly phase advanced under resonance cycles.

Circadian regulation of Fos-like expression in the brain of the blow fly Calliphora vicina

Expression of Fos-like immunoreactivity (FOS-lir) was examined in the brains of the blow fly Calliphora vicina for evidence of circadian regulation by photic stimuli. Fos-lir in various parts of the brain was investigated as a function of light and time of day. Immunohistochemistry demonstrated that photic stimuli have an inductive effect on c-fos expression in the various parts of the brain, but only in the neurons of the pars intercerebralis did the clear photic induction of c-fos expression occur at times when light was capable of phase-shifting circadian locomotor activity rhythms. This suggests that the c-fos gene may play a role in the photic pathway for circadian entrainment and that these neurons may be involved in the transduction of photic signals. Whether changes in c-fos expression are essential components of this pathway remains to be determined.