Dynamic Expression of Long Non-Coding RNAs (lncRNAs) in Adult Zebrafish

<div><p>Long non-coding RNAs (lncRNA) represent an assorted class of transcripts having little or no protein coding capacity and have recently gained importance for their function as regulators of gene expression. Molecular studies on lncRNA have uncovered multifaceted interactions with protein coding genes. It has been suggested that lncRNAs are an additional layer of regulatory switches involved in gene regulation during development and disease. LncRNAs expressing in specific tissues or cell types during adult stages can have potential roles in form, function, maintenance and repair of tissues and organs. We used RNA sequencing followed by computational analysis to identify tissue restricted lncRNA transcript signatures from five different tissues of adult zebrafish. The present study reports 442 predicted lncRNA transcripts from adult zebrafish tissues out of which 419 were novel lncRNA transcripts. Of these, 77 lncRNAs show predominant tissue restricted expression across the five major tissues investigated. Adult zebrafish brain expressed the largest number of tissue restricted lncRNA transcripts followed by cardiovascular tissue. We also validated the tissue restricted expression of a subset of lncRNAs using independent methods. Our data constitute a useful genomic resource towards understanding the expression of lncRNAs in various tissues in adult zebrafish. Our study is thus a starting point and opens a way towards discovering new molecular interactions of gene expression within the specific adult tissues in the context of maintenance of organ form and function.</p></div

Distribution of embryonic lncRNA transcripts in adult tissues of zebrafish.

<p>A. Clustered heat maps of 2,266 lncRNA transcripts obtained from Pauli et(H), liver (L), muscle (M), brain (Br) and blood (Bl) are represented. The color key represents the FPKM values in which grey color indicates the range from 0 to 10, light blue indicates the range from 11 to 100 and dark blue indicates 101 and above FPKM values for those with the highest expression. B. Enlarged section of the heat map depicting differential expression profile of 90 lncRNA transcripts expression across five tissues.</p

Real time assay for putative tissue restricted lncRNAs.

<p>Expression of candidate lncRNA transcripts was analyzed by semi quantitative RT-PCR in A) heart; B) liver; C) muscle; D) brain and E) blood tissues. A tissue specific protein coding marker gene viz <i>cmlc2</i> (heart); <i>tfr</i> (liver); <i>mdka</i> (brain); <i>murcb</i> (muscle) and <i>tal1</i> (blood) was used as standard control. See text for details on selection of protein coding marker genes. LncRNA transcripts investigated for a particular tissue type showed relatively predominant expression in the specific tissue when compared with other tissues.</p

RNA-sequencing data production and alignment results for tissue-specific Poly (A) reads.

<p>The total number of sequence reads obtained from the five zebrafish tissues using RNA sequencing is described. Mapped reads represent all transcripts that aligned back to the zebrafish reference genome (Zv9).</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

LncRNAs show tissue restricted expression patterns.

<p>Whole mount <i>in situ</i> hybridization of lncRNA transcripts. Shown are images with probes specific to the two indicated brain restricted lncRNAs. Arrow heads indicate the expression domains. A and B Anatomical cartoons of 24 hpf developing zebrafish embryo and adult zebrafish brain. C and D Expression of lncRNA transcript <i>lncBrHM_035.</i> (C) Dorsal view (anterior up) and lateral view (anterior to the left) showing expression in mid-hind brain boundary and hind brain of 24hpf zebrafish embryos. (D) Dorsal view (anterior up) of the adult zebrafish brain showing expression in regions of cerebellar crest (CC). E and F Expression of lncRNA transcript <i>lncBrM_002.</i> (E) Dorsal view (anterior up) and lateral view (anterior to the left) showing expression in fore-brain (FB), mid-hind brain boundary (MHB) and hind brain (HB) of 24hpf zebrafish embryos. (F) Dorsal view (anterior up) of the adult zebrafish brain showing expression in the regions of CC and a localized signal in eminentia granularis (EG). MB, mid brain; OB, olfactory bulb; Tel, telencephalon; Ha, habenula; Teo, optic tectum; MO, medulla oblongata.</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

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

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