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:The zebrafish pineal gland (epiphysis) is an autonomous clock organ. In addition to being a site of melatonin production, it contains photoreceptor cells and functions as a circadian clock pace maker, making zebrafish a useful model system to study the developmental control of expression of genes associated with melatonin synthesis and photodetection, and the circadian clock. Here we have used DNA microarray technology to study the zebrafish pineal transcriptome. Analysis of gene expression at five different developmental stages (three embryonic and two adult) has revealed a highly dynamic transcriptional profile, revealing many genes that are highly expressed in the pineal gland. Statistical analysis of the data based on Gene Ontology (GO) annotation indicates that many transcription factors and cell cycle genes are highly expressed during embryonic stages, whereas genes dedicated to visual system signal transduction are preferentially expressed in the adult. Furthermore, several genes were identified that exhibit day/night differences in expression. Our data provide a rich source of candidate genes for distinct functions at different stages of pineal gland development. Experiment Overall Design: Adults and embryos were kept under a 14-hr-light/10-hr-dark cycle. Pineal glands were isolated manually, guided by GFP fluorescence, from embryonic (3d, 5d, and 10d) and adult (3 month and 1-2 yr) transgenic zebrafish in which expression of the GFP gene is driven by the pineal-specific aanat2 promoter. For comparison, brain tissue from which the pineal gland and eyes had been removed was also collected (referred to as “brain”). Altogether, we collected 20 types of samples: five time points (3d, 5d, 10d, 3 mo, and 1-2 yr), two organs (pineal gland and brain), and two sampling times (day and night). For each type of sample, tissue was obtained and processed three to five times. Total RNA was prepared from each sample using the RNeasy Lipid Tissue Mini Kit (Qiagen) and biotin-labeled cDNA was generated using the Ovation Biotin system kit (NeuGen). The Affymetrix GeneChip® Zebrafish Genome Array was hybridized and processed using the standard Affymetrix protocol.
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: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 have circadian expression pattern 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, and transferred to constant darkness (DD) for tissue collection. Fish were anesthetized in 1.5 mM Tricane (Sigma), sacrificed by decapitation, and pineal glands were removed under a fluorescent dissecting microscope. Starting from circadian time (CT) 14, pineal glands were collected at 4-hr intervals for 48 hours (12 time points identified as CT 14, 18, 22, 2, 6, 10, 14b, 18b, 22b, 2b, 6b and 10b). Pools of 12 pineal glands were prepared at each time-point and total RNA was extracted using the RNeasy Lipid Tissue Mini Kit (QIAGEN), according to the manufacturer's instructions.