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:The rat pineal transcriptome is highly dynamic, with many hundreds of transcripts changing more than two-fold on a 24-hr basis, as revealed earlier using Affymetrix GeneChip analysis. The retina is evolutionally related to the pineal gland so these two tissues share many gene expression patterns. This study more completely characterizes the temporally dynamic transcriptomes of these tissues using RNA-Seq to capture information regarding alternative splicing, novel exons, unannotated mRNAs, non-coding RNAs, and coding transcripts not represented on the Affymetrix chips. We also identified transcripts that were selectively expressed in the pineal gland relative to other tissues by comparing pineal samples to a sample of pooled non-pineal tissues. The transcriptomes of the rat pineal gland and retina were sequenced using samples collected at 6 time points throughout a 24-hour cycle to identify rhythmically expressed transcripts. The transcriptomes of pools of mixed tissues collected at mid-day (ZT7) and mid-night (ZT19) were also sequenced for comparison to aid in determining pineal-enriched transcripts. Contributor: NISC, Comparative Sequencing Program
Project description:The rat pineal gland is a highly dynamic tissue with many hundreds of genes changing more than two-fold in a 24-hr daily rhythm as measured by Affymetrix GeneChip analysis. We sought to more completely understand this dynamic transcriptome using RNA-Seq in order to capture information regarding alternative splicing, novel exons, unannotated mRNAs, non-coding RNAs, etc. We also wished to identify transcripts that were selectively expressed in the pineal glands relative to other tissues. Toward this end we performed RNA-Seq on three types of samples; 1) a pool of pineal glands sampled at mid-day (ZT7); 2) a pool of pineal glands sampled at mid-night (ZT19); and a pool of 15 different tissues collected from 3 animals at mid-day (ZT7). Animals were housed in a 14:10 light:dark lighting cycle. PolyA-selected RNA was fragmented and sequenced on an Illumina GAII machine, yielding paired-end 51-mer reads.
Project description:NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures. NeuroD1 mRNA is highly abundant in the adult mammalian pineal gland and exhibits a developmental expression pattern similar to the retina. This is consistent with the common evolutionary origin of pinealocytes and retinal photoreceptors. Pinealocytes and retinal photoreceptors express a shared set of phototransduction genes and submammalian pinealocytes are photosensitive. In contrast to the retina, the pineal gland is a relatively homogeneous structure, composed 95% of pinealocytes. This makes the pineal gland a particularly useful model for understanding photoreceptor cell biology. The loss of NeuroD1 in the retina results in progressive photoreceptor degeneration and the molecular mechanisms underlying this retinal degeneration phenotype remain unknown. Similarly, the role that NeuroD1 plays in the pineal gland is unknown. To determine the function of NeuroD1 in the pineal gland and retina, a Cre/loxP recombination strategy was used to selectively target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, a transcription factor selectively expressed in the pineal gland and retina. Pineal and retinal tissues from two-month-old NeuroD1 cKO and control animals were used in microarray studies to identify candidate genes responsible for the photoreceptor degeneration phenotype. The Cre/loxP recombination strategy was used to target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout mice (NeuroD1floxed::Crx-Cre+/-). NeuroD1 floxed mice (NeuroD1floxed::Crx-Cre-/-) served as the controls. Pineal glands and retinas from two-month-old control and conditional knockout mice were collected at ZT6 and ZT20. 3 pools of 6 pineal glands per genotype and respective time of day were collected for each sample. Similarly, 3 pools of 6 retinas each were also collected. RNA from each pool was extracted and hybridized on the Affymetrix Mouse 430 2.0 array.
Project description:Biological processes are optimized by circadian and circannual biological timing systems. In vertebrates, the pineal gland plays an essential role in these systems by converting time into a hormonal signal, melatonin; in all vertebrates, circulating melatonin is elevated at night, independent of lifestyle. At night, sympathetic input to the pineal gland, originating from the circadian clock in the suprachiasmatic nucleus, releases norepinephrine. This adrenergic stimulation causes an elevation of cAMP, which is thought to regulate many of the dramatic changes in genes expression known to occur at night. In many aspects, the adrenergic/cAMP effects on gene expression can be recapitulated in primary organ culture. We have analyzed the rat pineal transcriptome at mid-day and mid-night to identify genes that exhibit night/day changes in expression. The pineal transcriptome was compared to that of other rat tissues processed in parallel. In addition, pineal glands were cultured in control conditions, or stimulated with norepinephrine, dibutyryl-cAMP (DBcAMP), or forskolin; the transcriptomes of these glands were then analyzed. Keywords: Time course (2 points) for in vivo pineal glands and various tissues; Treatment groups for cultured pineal glands
Project description:Biological processes are optimized by circadian and circannual biological timing systems. In vertebrates, the pineal gland plays an essential role in these systems by converting time into a hormonal signal, melatonin; in all vertebrates, circulating melatonin is elevated at night, independent of lifestyle. At night, sympathetic input to the pineal gland, originating from the circadian clock in the suprachiasmatic nucleus, releases norepinephrine. This adrenergic stimulation causes an elevation of cAMP, which is thought to regulate many of the dramatic changes in genes expression known to occur at night. In many aspects, the adrenergic/cAMP effects on gene expression can be recapitulated in primary organ culture. We have analyzed the rat pineal transcriptome at mid-day and mid-night to identify genes that exhibit night/day changes in expression. The pineal transcriptome was compared to that of other rat tissues processed in parallel. In addition, pineal glands were cultured in control conditions, or stimulated with norepinephrine, dibutyryl-cAMP (DBcAMP), or forskolin; the transcriptomes of these glands were then analyzed. Experiment Overall Design: Total RNA was extracted from various rat tissues, and from both in vivo and cultured rat pineal glands, for processing and hybridization to Affymetrix microarrays. Quadruplicates of pooled in vivo pineal glands were analyzed at each timepoint. Single day and night samples of retina, cortex, cerebellum, hypothalamus, liver, and heart were analyzed. Triplicates of control and treated cultured pineal glands were analyzed.
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:Pineal gland is a neuroendocrine gland located at the center of the brain. It protects the body from the effect of toxic compounds and regulates sleep-wake cycle, body temperature and sexual maturity through the secretion of melatonin. Abnormal functioning of pineal glands is known to be associated with Smith-Magenis syndrome, autism spectrum disorder, sleep disorders and Alzheimer’s disease. Characterization of pineal gland proteome will facilitate molecular level investigations on pathophysiological conditions underlying these diseases. We aimed to characterize the proteome of human pineal glands using a high resolution mass spectrometry- based approach. A total of 5,752 proteins were identified from human pineal glands in this study. Of these, 1,108 proteins contained signal peptide domain. We identified 2 novel proteins in this study, which are predicted by computational methods. In addition, a large number of proteins were uniquely identified in this study. A comprehensive list of proteins identified from human pineal glands will aid in unraveling the role of pineal glands in sleep disorders, neuropsychiatric and neurodegenerative diseases.
Project description:Biological processes are optimized by circadian and circannual biological timing systems. In vertebrates, the pineal gland plays an essential role in these systems by converting time into a hormonal signal, melatonin; in all vertebrates, circulating melatonin is elevated at night, independent of lifestyle. We have analyzed the rat pineal transcriptome at mid-day and mid-night to identify genes that exhibit night/day changes in expression. Experiment Overall Design: Rat pineal glands were obtained at mid-day and mid-night for RNA extraction and hybridization to Affymetrix microarrays. Triplicates of pooled pineal glands were analyzed at each timepoint. A similar set of samples was taken from a transgenic rat line (DN-Fra-2; Smith et al. (2001) Mol. Cell. Biol. 21, 3704-3713).
Project description:Biological processes are optimized by circadian and circannual biological timing systems. In vertebrates, the pineal gland plays an essential role in these systems by converting time into a hormonal signal, melatonin; in all vertebrates, circulating melatonin is elevated at night, independent of lifestyle. We have analyzed the rat pineal transcriptome at mid-day and mid-night to identify genes that exhibit night/day changes in expression. We have also used these data to characterize the non-rhythmic features of the transcriptome that set the pineal gland apart from other tissues by comparing them to the median expression in other rat tissues as found in the Genomics Institute of the Novartis Research Foundation (GNF), Entrez Gene Expression Omnibus (GEO) dataset GDS589. Experiment Overall Design: Rat pineal glands were obtained at mid-day and mid-night for RNA extraction and hybridization to Affymetrix microarrays. Triplicates of pooled pineal glands were analyzed at each timepoint.