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

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

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

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

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

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used