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Next Generation Sequencing Study of Circadian Changes in Transcriptome of Zebrafish Pineal Gland and Eye

ABSTRACT: Purpose: We performed an NGS study on the circadian changes in zebrafish eye and pineal gland transcriptome in order to elucidate novel and conserved elements in the circadian clock. Methods: Poly-A selected RNA from zebrafish eyes and pineaal glands of animals eutanized at 2 timepoints (Mid-Day, Midnight) was deep sequenced, using Illumina HiSeq2000. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2. Results: We discover a variety of genes that show circadian activivty in both eye and pineal gland of Zebrafish. Conclusions: Our study represents part of a comparative analysis of retinal(eye) and pineal gland transcriptome of several species, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

ORGANISM(S): Danio rerio

PROVIDER: GSE99285 | GEO | 2018/02/01

REPOSITORIES: GEO

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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.

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Next Generation Sequencing Study of Circadian Changes in Transcriptome of Zebrafish Pineal Gland and Eye

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