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Next Generation Sequencing Study of Circadian Changes in Transcriptome of Rhesus Macaque Pineal Gland and Retina

ABSTRACT: Purpose: We performed an NGS study on the circadian changes in Rhesus Macaque retina and pineal gland transcriptome in order to elucidate novel elements in the primate circadian clock. Methods: Total RNA from retina and pineaal glands of Rhesus Macauqes euthanized at 4 timepoints (Dawn, Day, Dusk and Night) was deep sequenced, in triplicate, using Illumina HiSeq2000. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2. Results: We discover a variety of novel genes that show circadian activivty in both retina and pineal gland of the Rhesus Macaque. A particular set of genes involved in the production of N-acetyltryptamine in Rhesus Macaque retina appears to be responsible for the circadian oscillations of the concentrations of N-acetyltriptamine in the plasma, thereby accentuating N-acetyltriptamine as a retina derived signaling molecule in primates. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, 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): Macaca mulatta

PROVIDER: GSE78165 | GEO | 2017/06/01

SECONDARY ACCESSION(S): PRJNA312819

REPOSITORIES: GEO

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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.

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Next Generation Sequencing Study of Circadian Changes in Transcriptome of Rhesus Macaque Pineal Gland and Retina

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