Unknown,Transcriptomics,Genomics,Proteomics

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CAGE-seq of zebrafish cells extracted at the 14 somite stage and segregated by cell cycle dynamics (e.g. fast - green vs slow - red)

ABSTRACT: The core-promoter, a stretch of DNA surrounding the transcription-start-site (TSS), is a major integration-point for regulatory-signals controlling gene-transcription. Cellular-differentiation is marked by divergence in transcriptional-repertoire and cell- cycling behaviour between cells of different fates. The role promoter-associated gene- regulatory-networks play in development-associated transitions in cell-cycle- dynamics is poorly understood. This study demonstrates in a vertebrate embryo, how core-promoter variations define transcriptional-output in cells transitioning from a proliferative to cell-lineage specifying phenotype. Assessment of cell-proliferation across zebrafish embryo-development, using the FUCCI-transgenic cell-cycle- phase marker, revealed a spatial and lineage-specific separation in cell-cycling behaviour. To investigate the role differential promoter-usage plays in this process, cap-analysis-of-gene-expression (CAGE) was performed on cells segregated by cycling-dynamics.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Danio rerio

SUBMITTER: Joseph Wragg

PROVIDER: E-MTAB-8795 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This project aimed at identifying developmental stage specific transcript profiles for catecholaminergic neurons in embryos and early larvae of zebrafish (Danio rerio). Catecholaminergic neurons were labeled using transgenic zebrafish strains to drive expression of GFP. At stages 24, 36, 72 and 96 hrs post fertilization, embryos were dissociated and GFP expressing cells sorted by FACS. Isolated RNAs were processed using either polyA selection and libray generation or NanoCAGE. This is the first effort to determine stage specific mRNA profiles of catecholaminergic neurons in zebrafish. Catecholaminergic neurons were labeled by four different strategies: (1) 24 hrs old embryos: we used the ETvmat2:GFP transgenic line (Wen et al. 2007). Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish. Dev Biol. 2008 Vol 314 p84-92) which at this early stage labels catecholaminergic neurons in posterior tuberculum and locus coeruleus; (2) 24 hrs old embryos: we used Tg(otpb.A:egfp)zc48 transgenic line (Fujimoto et al. Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification. Dev Biol 2011, Vol 352, p393–404) which at this stage label ventral diencephalic dopaminergic neurons and some preoptic neurons. (3) For 72 and 96 hrs old zebrafish larvae we used a th:GFP BAC transgenic lines that labels catecholaminergic neurons (Tay et al., Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems. Nat Comms 2011 Vol 2, 171; also: T. Leng and W. Driever, unpublished). (4) for the 36 and 48 hrs old zebrafish larvae we used a th:Gal4VP16 driver and UAS:EGFP responder transgenic line system to label catecholaminergic cells (Fernandes et al., Deep brain photoreceptors control light-seeking behavior in zebrafish larvae. Curr Biol. 2012 Vol 22 DOI 10.1016/j.cub.2012.08.016). We used the different transgenic lines, because lines (3) and (4) do not efficiently label catecholaminergic neurons at early stages, while lines (1) and (2) also have GFP expression in several other non-catecholaminergic populations at later stages of development. Embryos were dissociated and catecholaminergic neurons were FACS sorted from GFP-tagged zebrafish (Manoli and Driever, 2012, Cold Spring Harbor Protoc. DOI 10.1101/pdb.prot069633). RNA was either processed for NanoCAGE, or mRNA was isolated and amplified. cDNA was sequenced by Illumina technique. This data submission is a series of data files consisting of three independent experiments with diffrent RNA-Seq depth: Samples 1-4 (NanoCage): Samples 5-8 (RNA-Seq high read numbers), and SAmples 9-12 (RNA-Seq low read numbers).

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CAGE-seq of zebrafish cells extracted at the 14 somite stage and segregated by cell cycle dynamics (e.g. fast - green vs slow - red)

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