Project description:Purpose: We aimed to investigate important miRNA events and to define specific miRNA expression signature underlying the follicle activation of zebrafish. Methods: By using small and regular RNA-sequencing, we performed transcriptomic analyses of PG (primary growth stage I; inactive) and PV (pre-vitellogenic stage II; activated) follicles to decipher important miRNA and gene events underlying follicle activation of zebrafish. We identified differentially expressed miRNAs for subsequent qPCR validation and miRNA::target gene prediction. Interaction of candidate miRNA:: target gene pairs were further validated by luciferase reporter assay. Global gene networks involved during PG-to-PV transition were also assessed by Gene Ontology as well as KEGG pathway analyses. Results and Conclusion: Our expression results indicated that PG follicles can be well differentiated from PV follicles by simply using a specific 13-miRNA expression signature (let-7a, -7b, -7c-5p, -7d-5p, -7h, -7i; miR-21, -23a, -27c-3p, -107a-3p, -125b-5p, -145-3p, -202-5p). Besides, we validated interactions of let-7i::atg4a, miR-202-5p::c23h20orf24 and miR-144::ybx1 by luciferase reporter assay. Purpose: we aimed to investigate important miRNA events and to define specific miRNA expression signature underlying the follicle activation of zebrafish
Project description:Low temperatures may cause severe growth inhibition and mortality in fish. In order to understand the mechanism of cold tolerance, a transgenic zebrafish Tg (smyd1:m3ck) model was established to study the effect of energy homeostasis during cold stress. The muscle-specific promoter Smyd1 was used to express the carp muscle form III of creatine kinase (M3-CK), which maintained enzymatic activity at a relatively low temperature, in zebrafish skeletal muscle. In situ hybridization showed that M3-CK was expressed strongly in the skeletal muscle. When exposed to 13°C, Tg (smyd1:m3ck) fish maintained their swimming behavior, while the wild-type could not. Energy measurements showed that the concentration of ATP increased in Tg (smyd1:m3ck) versus wild-type fish at 28°C. After 2 h at 13°C, ATP concentrations were 2.16-fold higher in Tg (smyd1:m3ck) than in wild-type (P < 0.05). At 13°C, the ATP concentration in Tg (smyd1:m3ck) fish and wild-type fish was 63.3% and 20.0%, respectively, of that in wild-type fish at 28°C. Microarray analysis revealed differential expression of 1249 transcripts in Tg (smyd1:m3ck) versus wild-type fish under cold stress. Biological processes that were significantly overrepresented in this group included circadian rhythm, energy metabolism, lipid transport, and metabolism. These results are clues to understanding the mechanisms underlying temperature acclimation in fish.
Project description:Compared to other fish models, miRNAs are currently most extensively studied and identified in zebrafish. Approximately 415 dre-miRNAs have been identified and several articles have studied some aspect of miRNA function in zebrafish such as their role in basic development and in disease pathways. However, this field of research is in its infancy and the function of several dre-miRNAs, as well as their tissue-specific expression profile, are yet to be defined. In this study, the liver and gut were dissected (wildtype/untreated fish), total and small RNA were extracted, mRNA and miRNA libraries constructed and subjected to high throughput sequencing (HTS) using standard approaches. We carried out differential expression (DE) analysis and compared liver miRNA expression to gut using established bioinformatics pipelines. Through bioinformatics analysis, known and putative novel miRNAs were identified. Finally, we constructed a “miRNA matrix” that connects both total RNA-Seq and miRNA-Seq.
Project description:Compared to other fish models, miRNAs are currently most extensively studied and identified in zebrafish. Approximately 415 dre-miRNAs have been identified and several articles have studied some aspect of miRNA function in zebrafish such as their role in basic development and in disease pathways. However, this field of research is in its infancy and the function of several dre-miRNAs, as well as their tissue-specific expression profile, are yet to be defined. In this study, the liver and gut were dissected (wildtype/untreated fish), total and small RNA were extracted, mRNA and miRNA libraries constructed and subjected to high throughput sequencing (HTS) using standard approaches. We carried out differential expression (DE) analysis and compared liver miRNA expression to gut using established bioinformatics pipelines. Through bioinformatics analysis, known and putative novel miRNAs were identified. Finally, we constructed a “miRNA matrix” that connects both total RNA-Seq and miRNA-Seq.