Project description:Light has a strong effect on whole organism physiology, such as the circadian rhythms that are phase delayed and advanced by light given at early and late subjective night, respectively. Despite the importance of the phase-dependent light responses, little is known about the underlying molecular mechanism. We performed a comprehensive analysis of genes induced by light in a phase-dependent manner in the chicken pineal gland, an organ that represents a unique vertebrate clock system harboring intrinsic light sensitivity. Newborn chicks were entrained to 12-h light/12-h dark cycle for 7 days then transferred to constant darkness for a day to be exposed to light for 1 h from CT (circadian time) 6 (representing subjective day), CT14 (early subjective night) or CT22 (late subjective night). Control animals were kept in the dark without light pulse. The pineal glands were isolated at the end of the 1-h light pulse for gene expression analysis by Affymetrix GeneChip. Each condition contains 2 biological samples.
Project description:Light is the primary environmental cue in resetting the phase of circadian pacemaker in vertebrates. In birds, the effect of light is partly mediated by modulating the levels of circadian genes in the pineal gland. To further elucidate the mechanism by which light resets the circadian clock, we studied gene expression in the chicken pineal gland under acutely extended light period.
Project description:Light is the primary environmental cue in resetting the phase of circadian pacemaker in vertebrates. In birds, the effect of light is partly mediated by modulating the levels of circadian genes in the pineal gland. To further elucidate the mechanism by which light resets the circadian clock, we studied gene expression in the chicken pineal gland under acutely extended light period. Three paradigms of treatments were used in this study. For each paradigm, chicks were assigned at random to control treatment (control groups) or light treatment (light groups). All birds in control groups were given 12 h light and 12 h dark period (LD 12:12). Light-on time is referred to as Zeitgeber Time 0 (ZT0). In paradigm 1, birds in the light group (n =25 for each of the groups in each paradigm) were acclimated to LD 12:12 for one week in the same light scheme as were the control birds, then exposed to light for 2 h during the subjective late night (ZT22 to ZT24) on the last day. All birds (including the controls) were sacrificed at ZT0. Pineal glands were dissected and 5-6 pineal glands were pooled for the preparation of one RNA sample. In paradigm 2, birds in the light group were acclimated as in paradigm 1 for one week, then exposed to light for 2 h during the early subjective night (ZT12 to ZT14) on the last day. All birds (including controls) were sacrificed at ZT14. The pineal glands were also pooled as before. In paradigm 3, birds in the light group (n = 25) were kept in LD 15:9 cycle all the time, and all birds (including controls) were sacrificed at ZT14. Similarly, 5-6 pineal glands in the same treatment were pooled.
Project description:Light has a strong effect on whole organism physiology, such as the circadian rhythms that are phase delayed and advanced by light given at early and late subjective night, respectively. Despite the importance of the phase-dependent light responses, little is known about the underlying molecular mechanism. We performed a comprehensive analysis of genes induced by light in a phase-dependent manner in the chicken pineal gland, an organ that represents a unique vertebrate clock system harboring intrinsic light sensitivity.
Project description:Using mass spectrometry based proteomics and dedicated analysis tools, we identify proteins expressed in the human pineal gland and analyze systematically their variation with time of the throughout the day, and, compare these changes in the pineal proteome between control specimens and donors diagnosed with ASD.
Project description:This study determines pineal gland gene expression levels in the NeuroD1 knockout mouse at postnatal day zero. Comparison was performed against pineal gland gene expression levels in 129 wildtype mice also disected at P0. Keywords: Comparison of wildtype versus transgenic pineal gland gene expression
Project description:Microarray data allowed detection of genes that are induced by light in the zebrafish pineal gland Adult (0.5-1.5 years old) transgenic zebrafish, Tg(aanat2:EGFP)Y8, which express enhanced green fluorescent protein (EGFP) in the pineal gland under the control of the aanat2 regulatory regions, were used. Fish were raised under 12-hr light:12-hr dark (LD) cycles, in a temperature controlled room. Fish were transferred to constant darkness (DD) at the end of the day prior to the experiment. Fish were exposed to a 1-hr light pulse (light intensity of 12 W/m2) prior to sampling (light treatment) or kept under constant darkness for control (dark treatment). The tissues were collected from light- and dark-treated fish at 6 time points with 4-hr intervals throughout one daily cycle, corresponding to CT2, 6, 10, 14, 18 and 22. Fish were anesthetized in 1.5 mM Tricane (Sigma), sacrificed by decapitation, and pineal glands were removed under a fluorescent dissecting microscope. Pools of 12 pineal glands were prepared at each condition and total RNA was extracted using the RNeasy Lipid Tissue Mini Kit (QIAGEN), according to the manufacturer's instructions.
Project description:Microarray data allowed detection of genes that are highly expressed in the pineal gland. Experiment Overall Design: Adult Tg(aanat2:EGFP)Y8 transgenic zebrafish in which EGFP marks the pineal gland were used for RNA extraction and hybridization on Affymetrix microarrays. Fish were anesthetized in 1.5 mM Tricane (Sigma) and sacrificed by decapitation, and pineal glands were removed under a fluorescent dissecting microscope. Since the pineal gland is a clock-containing organ and levels of certain transcripts may vary throughout the circadian cycle, glands were collected throughout the 24-hr cycle at 4 hr intervals. During the 24-hr cycle the fish were either maintained in a 12-h light/12-h dark cycle (LD) or kept in constant darkness (DD). 12 pineal glands were collected and pooled at each time point at each light condition, and total RNA was extracted (RNeasy, Qiagen).
Project description:Chick pinealocytes exhibit all the characteristics of a complete circadian system, comprising photoreceptive inputs, molecular clockworks and an easily measured rhythmic output, melatonin biosynthesis. We used microarray analysis to investigate the expression of approximately 8000 genes within cultured pinealocytes subjected to both LD and DD cycles. We report that a reduced subset of genes were rhythmically expressed in vitro compared to those previously published in vivo, and that gene expression rhythms were lower in amplitude, although the functional distribution of the rhythmic transcriptome was largely similar. We also investigated the effects of 6-hour pulses of light or of norepinephrine on gene expression in free-running cultures during both subjective day and night. As expected, both light and norepinephrine inhibited melatonin production; however, the two treatments differentially up- or down-regulated specific sets of genes in a fashion that was dependent upon time of day. Our combined approach of utilizing a time of day study and a light/NE pulse microarray experiment allowed us to identify novel genes linking clock input to clock function within the pineal. We identified approximately 30 rhythmic, light-responsive, NE-insensitive genes with no previously known clock function, which may play a role in circadian regulation of the pineal. These are candidates for future functional genomics experiments to elucidate their potential role in pineal physiology. Keywords: circadian; avian; pineal; light; norepinephrine