The embryonic pineal gland of the chicken as a model for experimental jet lag.

The circadian clock in the chicken pineal model develops before hatching, at around the 17th embryonic day (ED17). By this stage, it runs in synchrony with environmental cues. To address if phase resetting mechanisms are comparable to those of post-hatched chicken, we investigated ED19 stage chicken embryos under 12h light:12h dark (LD), under constant darkness (DD), or under acute 4h phase delay of the LD condition (LD+4). The 24h mRNA-expression patterns of clock gene clock and of clock controlled genes Aanat and hiomt were analyzed with qRT-PCR. Under DD the rhythm of Aanat did not change significantly, however the 24h pattern of hiomt was altered. Clock shows a delayed response to DD with a phase-shift in its rhythm. After the first cycle under LD+4 conditions, the 24h patterns of Aa-nat and hiomt mRNA-s were phase delayed. Clock showed both acute and delayed changes in response to LD+4. These results show that the embryonic chicken pineal gland has a fully functioning clock mechanism, and that it is a good model for phase-change experiments. In addition it demonstrates that only one cycle of altered light schedule is sufficient to trigger changes within the molecular clock mechanisms of the chicken embryonic pineal model.

Altered expression patterns of clock gene mRNAs and clock proteins in human skin tumors.

The majority of our genes may be regulated in a daily rhythm, including the genes for cell cycle control. Epidemiological and genetic evidences suggest that disruption of circadian timing mechanisms makes our cells more vulnerable to cancer formation. The aim of this study was to investigate the relationship between expression patterns of circadian clock genes (period homolog (per)1, per2, clock, and cry1) and tumor development by analyzing human skin biopsies of malignant melanoma and nonmalignant naevus tumors. We found that mRNA levels and nuclear immunopositivity for the investigated clock genes were reduced by 30-60 % in both melanoma and in naevus biopsies if compared with adjacent nontumorous samples. The alterations in melanoma presented significant associations with clinicopathological characteristics (e.g., Breslow thickness). Contrary to previous reports, the moderate decrease of per1 expression seen in malignant tissues could not be linked to malignant transformation itself; rather, it reflects only the alterations in tissue composition. In turn, clock expression was upregulated in nontumorous cells of melanoma biopsies but not in melanoma cells or naevus cells. As this gene (clock) is closely related to cellular metabolism, our data suggest its role in the impaired regulation of metabolism in malignant tumors. Our results present the first clinical evidence for a possible link between circadian clock genes and human skin tumorigenesis.

Expression pattern of clock under acute phase-delay of the light/dark cycle in the chicken pineal model.

Shift workers have a higher risk of metabolic syndrome, a condition that also develops in mice carrying mutation in their circadian clock gene clock. To collect more data on the transcriptional changes of clock under phase-shifted light/dark LD conditions, we examined the 24h patterns of clock mRNA expression in vivo and in vitro in chickens exposed acutely to a reversed LD (DL) cycle. Under controlled LD conditions (lights on at 6:00, lights off at 20:00), clock mRNA expression peaked in vivo at 2:00 (Zeitgeber Time 20, ZT20) and in vitro at 22:00 (ZT16). Even higher mRNA contents were measured in the first cycle of in vivo DL conditions between 22:00 and 6:00 (lights at night), but in the second cycle by 2:00, lower mRNA contents were detected than the control peak values seen at this time point. Furthermore, no alterations were found in vitro in clock mRNA content during the first 12h of DL conditions (lights at night). The differences seen between the first and the second DL cycles in vivo and between the in vivo and in vitro data for the first DL cycle support the idea that neurohumoral signals perturbed by a phase-delayed light-dark cycle may also play a role in the in vivo rapid transcriptional resetting of the circadian clock in the chicken pineal model.

Circadian expression of clock genes clock and Cry1 in the embryonic chicken pineal gland.

Clock and Cry1 expression were examined in the pineal gland of chicken embryos incubated under constant darkness from embryonic day (ED) 0. From ED13, Clock and Cry1 mRNA levels showed episodic alterations. After ED17, circadian pattern of clock gene expression was seen both in vivo and in vitro. Our results support the idea that rhythmic environmental factors are not necessary for the generation of circadian patterns of clock gene expression during development.

Expression of Cry2 in the chicken pineal gland: effects of changes in light-dark conditions.

Pineal expression of Cry2 mRNA has been examined in chickens under normal (LD) and reversed (DL) light-dark conditions. In vivo the peak of Cry2 mRNA content at late subjective day under LD diminished after switching to a DL schedule. In vitro, Cry2 mRNA levels showed a steady decrease during light exposure at subjective night. Our data show that light-sensitive clock components in the pinealocytes may be involved in the repression of Cry2 transcription at night, which may contribute to resetting the phase of the clock within 24 h.