zflncRNApedia: A Comprehensive Online Resource for Zebrafish Long Non-Coding RNAs

<div><p>Recent transcriptome annotation using deep sequencing approaches have annotated a large number of long non-coding RNAs in zebrafish, a popular model organism for human diseases. These studies characterized lncRNAs in critical developmental stages as well as adult tissues. Each of the studies has uncovered a distinct set of lncRNAs, with minor overlaps. The availability of the raw RNA-Seq datasets in public domain encompassing critical developmental time-points and adult tissues provides us with a unique opportunity to understand the spatiotemporal expression patterns of lncRNAs. In the present report, we created a catalog of lncRNAs in zebrafish, derived largely from the three annotation sets, as well as manual curation of literature to compile a total of 2,267 lncRNA transcripts in zebrafish. The lncRNAs were further classified based on the genomic context and relationship with protein coding gene neighbors into 4 categories. Analysis revealed a total of 86 intronic, 309 promoter associated, 485 overlapping and 1,386 lincRNAs. We created a comprehensive resource which houses the annotation of lncRNAs as well as associated information including expression levels, promoter epigenetic marks, genomic variants and retroviral insertion mutants. The resource also hosts a genome browser where the datasets could be browsed in the genome context. To the best of our knowledge, this is the first comprehensive resource providing a unified catalog of lncRNAs in zebrafish. The resource is freely available at URL: <a href="http://genome.igib.res.in/zflncRNApedia" target="_blank">http://genome.igib.res.in/zflncRNApedia</a></p></div

Workflow detailing data curation and methodologies involved in building the resource.

<p>Workflow detailing data curation and methodologies involved in building the resource.</p

Matrix reporting sample count of various datasets used across different developmental time-points.

<p>Matrix reporting sample count of various datasets used across different developmental time-points.</p

Chamber Specific Gene Expression Landscape of the Zebrafish Heart

<div><p>The organization of structure and function of cardiac chambers in vertebrates is defined by chamber-specific distinct gene expression. This peculiarity and uniqueness of the genetic signatures demonstrates functional resolution attributed to the different chambers of the heart. Altered expression of the cardiac chamber genes can lead to individual chamber related dysfunctions and disease patho-physiologies. Information on transcriptional repertoire of cardiac compartments is important to understand the spectrum of chamber specific anomalies. We have carried out a genome wide transcriptome profiling study of the three cardiac chambers in the zebrafish heart using RNA sequencing. We have captured the gene expression patterns of 13,396 protein coding genes in the three cardiac chambers—atrium, ventricle and bulbus arteriosus. Of these, 7,260 known protein coding genes are highly expressed (≥10 FPKM) in the zebrafish heart. Thus, this study represents nearly an all-inclusive information on the zebrafish cardiac transcriptome. In this study, a total of 96 differentially expressed genes across the three cardiac chambers in zebrafish were identified. The atrium, ventricle and bulbus arteriosus displayed 20, 32 and 44 uniquely expressing genes respectively. We validated the expression of predicted chamber-restricted genes using independent semi-quantitative and qualitative experimental techniques. In addition, we identified 23 putative novel protein coding genes that are specifically restricted to the ventricle and not in the atrium or bulbus arteriosus. In our knowledge, these 23 novel genes have either not been investigated in detail or are sparsely studied. The transcriptome identified in this study includes 68 differentially expressing zebrafish cardiac chamber genes that have a human ortholog. We also carried out spatiotemporal gene expression profiling of the 96 differentially expressed genes throughout the three cardiac chambers in 11 developmental stages and 6 tissue types of zebrafish. We hypothesize that clustering the differentially expressed genes with both known and unknown functions will deliver detailed insights on fundamental gene networks that are important for the development and specification of the cardiac chambers. It is also postulated that this transcriptome atlas will help utilize zebrafish in a better way as a model for studying cardiac development and to explore functional role of gene networks in cardiac disease pathogenesis.</p></div

Data workflow and analysis summary.

<p>An overview of experimental and data analysis workflow adopted in the study for identification of known and putative novel cardiac chamber-restricted protein coding genes. (B) Venn diagram representing the total number of known protein coding genes (7,260) identified with expression level > = 10 FPKM across the three cardiac chambers. (C) A heat map representing expression pattern of 7,260 RefSeq protein coding genes with expression level > = 10 FPKM in the three cardiac chambers. The colour key represents transcripts in the range of 0 for transcripts with least expression to 15 for transcripts with maximum expression.</p

Quantitative Real time PCR based validation of chamber-restricted candidate genes.

<p>Atrium-restricted genes (a), (b) and (c). Ventricle-restricted genes (d), (e), (f) and (g). Bulbus arteriosus-restricted genes (h) and (i). See text for detailed information.</p

Differential expression of genes in the three cardiac chambers.

<p>The heat map represents expression profile of 96 cardiac chamber-restricted genes. The first column in the adjoining table lists the corresponding zebrafish gene names which are colour coded to highlight those with mutant phenotypes. Genes marked with green colour signify those which have been identified with heart related mutant phenotypes. The second column lists the human orthologs known for these genes. Genes marked with asterisks (*) represent those mentioned in the text (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147823#sec012" target="_blank">Results</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147823#sec023" target="_blank">Discussion</a>). Colour coding for the human orthologs highlights those which show disease associations. Orthologs marked with red colour are associated with cardiac disorders. The colour key represents transcripts in the range of 0 for transcripts with least expression to 15 for transcripts with maximum expression.</p

Developmental and adult tissue expression profile of the putative novel protein coding genes identified in the ventricular chamber.

<p>The heat maps represent spatiotemporal expression profile of 23 ventricle-restricted putative novel protein coding genes across 11 zebrafish developmental stages and 6 adult tissue types. The gene expression data for the embryonic and larval developmental stages were obtained from previous published studies (Ulitsky et al, 2011; Pauli et al, 2012) that utilized whole embryos or larvae. In contrast our dataset was obtained exclusively from the adult heart tissue. Thus, the expression profiles of the novel ventricular-restricted genes may not reflect in parallel to the early ventricular developmental hallmarks. The colour key represents transcripts in the range of 0 for genes with least expression to 5 for transcripts with maximum expression.</p

Developmental and adult tissue expression profiles of differentially expressed genes in the three cardiac chambers.

<p>The heat maps represent spatiotemporal expression profile of 96 chamber-restricted genes across 11 zebrafish developmental stages and 6 adult tissue types. The colour key represents transcripts in the range of 0 for genes with least expression to 10 for transcripts with maximum expression. Genes marked with asterisks (*) represent those mentioned in the text (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147823#sec012" target="_blank">Results</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147823#sec023" target="_blank">Discussion</a>).</p

Summary of the RNA sequencing data generation and alignment.

<p>Total number of sequence reads obtained from the three cardiac chambers using RNA sequencing is described. Mapped reads refer to and include all transcripts that aligned back to the zebrafish reference genome (Zv9). The total number of uniquely expressing known protein coding genes as well as <i>de novo</i> transcripts are mentioned.</p