Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of zebrafish pineal gland and brain tissue from embryonic and adult fish

ABSTRACT: 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.

ORGANISM(S): Danio rerio

SUBMITTER: Reiko Toyama

PROVIDER: E-GEOD-13371 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The vertebrate pineal gland is dedicated to the production of the hormone melatonin, which increases at night to influence circadian and seasonal rhythms. This increase is associated with dramatic changes in the pineal transcriptome. Here, single-cell analysis of the rat pineal transcriptome was approached by sequencing mRNA from ~17,000 individual pineal cells, with the goals of profiling the cells that comprise the pineal gland and examining the proposal that there are two distinct populations of pinealocytes differentiated by the expression of Asmt, which encodes the enzyme that converts N-acetylserotonin to melatonin. In addition, this analysis provides evidence of cell-specific time-of-day dependent changes in gene expression. Nine transcriptomically distinct cell types were identified: ~90% were classified as melatonin-producing α- and β-pinealocytes (1:19 ratio). Non-pinealocytes included three astrocyte subtypes, two microglia subtypes, vascular and leptomeningeal cells, and endothelial cells. α-Pinealocytes were distinguished from β-pinealocytes by ~3-fold higher levels of Asmt transcripts. In addition, α-pinealocytes have transcriptomic differences that likely enhance melatonin formation by increasing the availability of the Asmt cofactor S-adenosylmethionine, resulting from increased production of a precursor of S-adenosylmethionine, ATP. These transcriptomic differences include ~2-fold higher levels of the ATP-generating oxidative phosphorylation transcriptome and ~8-fold lower levels of the ribosome transcriptome, which is expected to reduce the consumption of ATP by protein synthesis. These findings suggest that α-pinealocytes have a specialized role in the pineal gland: efficiently O-methylating the N-acetylserotonin produced and released by β-pinealocytes, thereby improving the overall efficiency of melatonin synthesis. We have also identified transcriptomic changes that occur between night and day in seven cell types, the majority of which occur in β-pinealocytes and to a lesser degree in α-pinealocytes; many of these changes were mimicked by adrenergic stimulation with isoproterenol. The cellular heterogeneity of the pineal gland as revealed by this study provides a new framework for understanding pineal cell biology at single-cell resolution.

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Transcription profiling of zebrafish pineal gland and brain tissue from embryonic and adult fish

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