Gen-Hwa Lin

Differential expression patterns of growth-related microRNAs in the skeletal muscle of Nile tilapia (Oreochromis niloticus)1

MicroRNA (miRNA) are a class of small, single-stranded, non-coding RNA that regulate mRNA expression at the post-transcriptional level and play important roles in many fundamental biological processes. There is emerging evidence that miRNA are critical regulators of widespread cellular functions, such as differentiation, proliferation, and migration. At present, little is known about miRNA expression profiles related to skeletal muscle growth in aquatic organisms. This study aimed to investigate the phenotypic variation in the body growth of the Nile tilapia (Oreochromis niloticus) and to identify and quantify the differential expression levels of selected growth-related transcriptomic miRNA in the skeletal muscle of this fish. To this end, we performed next-generation sequencing to define the full miRNA transcriptome in muscle tissue from Nile tilapia and to detect differentially expressed miRNA between 2 strains of Nile tilapia. These tilapia strains exhibited significant (P < 0.05) phenotypic variation with respect to growth-related traits (body length and BW), mitochondrial DNA (mtDNA) haplotype diversity, and the differential expression of selected growth-related genes. The results obtained from the transcriptome analysis and real-time quantitative reverse transcription PCR (qRT-PCR) revealed significant differences in miRNA expression between fast-growing and control strains of tilapia. Digital gene expression (DGE) profiling was performed based on the obtained read abundance, and we identified down-regulated miRNA, including let-7j, miR-140, miR-192, miR-204, miR-218a, miR-218b, miR-301c, and miR-460, and up-regulated miRNA, including let-7b, let-7c, miR-133, miR-152, miR-15a, miR-193a, miR-30b, and miR-34, associated with body growth in tilapia. These results were further validated using real-time qRT-PCR and microarray profiling. In summary, the up- and down-regulation of miRNA involved in the GH/IGF-1 axis signaling pathway suggests that the differential expression levels of growth-related miRNA may serve as molecular markers that are predictive of specific functional and diagnostic implications. The obtained data on genetic polymorphisms in miRNA-target interactions are particularly useful for Nile tilapia breeding programs.

Progranulin A-mediated MET signaling is essential for liver morphogenesis in zebrafish.

The mechanism that regulates embryonic liver morphogenesis remains elusive. Progranulin (PGRN) is postulated to play a critical role in regulating pathological liver growth. Nevertheless, the exact regulatory mechanism of PGRN in relation to its functional role in embryonic liver development remains to be elucidated. In our study, the knockdown of progranulin A (GrnA), an orthologue of mammalian PGRN, using antisense morpholinos resulted in impaired liver morphogenesis in zebrafish (Danio rerio). The vital role of GrnA in hepatic outgrowth and not in liver bud formation was further confirmed using whole-mount in situ hybridization markers. In addition, a GrnA deficiency was also found to be associated with the deregulation of MET-related genes in the neonatal liver using a microarray analysis. In contrast, the decrease in liver size that was observed in grnA morphants was avoided when ectopic MET expression was produced by co-injecting met mRNA and grnA morpholinos. This phenomenon suggests that GrnA might play a role in liver growth regulation via MET signaling. Furthermore, our study has shown that GrnA positively modulates hepatic MET expression both in vivo and in vitro. Therefore, our data have indicated that GrnA plays a vital role in embryonic liver morphogenesis in zebrafish. As a result, a novel link between PGRN and MET signaling is proposed.

RIG-I specifically mediates group II type I IFN activation in nervous necrosis virus infected zebrafish cells

The type I interferon (IFN) response has been shown to be crucial for the survival of zebrafish larvae infected with nervous necrosis virus (NNV). Teleost type I IFNs can be divided into two groups, based on their cysteine content. While teleost group I IFNs have been extensively studied in terms of their regulation and anti-viral properties, the characteristics of teleost group II IFNs have been relatively unexplored. In this study, we describe the mechanism by which group II IFNs are activated in response to NNV infection in a zebrafish cell line, by focusing on the relationship between type I IFNs and pattern recognition receptors. Expression profile analysis of infected cells by microarray and qPCR revealed signaling activation of two pattern recognition receptors (PRRs): RIG-I like receptors (RLRs) and MyD88-dependent Toll-like receptors (TLRs). Knockdown of retinoic acid-inducible gene I (RIG-I) specifically repressed induction of group II IFNs (IFNϕ2, IFNϕ3) by NNV infection. Furthermore, Ingenuity Pathway Analysis (IPA) was used to demonstrate that RIG-I knockdown results in down-regulation of the inflammatory response in NNV-infected cells. Taken together, our results indicate that RIG-I plays an essential role in zebrafish group II type I IFN induction and the inflammatory response to NNV infection.

Progranulin regulates zebrafish muscle growth and regeneration through maintaining the pool of myogenic progenitor cells

Myogenic progenitor cell (MPC) is responsible for postembryonic muscle growth and regeneration. Progranulin (PGRN) is a pluripotent growth factor that is correlated with neuromuscular disease, which is characterised by denervation, leading to muscle atrophy with an abnormal quantity and functional ability of MPC. However, the role of PGRN in MPC biology has yet to be elucidated. Here, we show that knockdown of zebrafish progranulin A (GrnA) resulted in a reduced number of MPC and impaired muscle growth. The decreased number of Pax7-positive MPCs could be restored by the ectopic expression of GrnA or MET. We further confirmed the requirement of GrnA in MPC activation during muscle regeneration by knockdown and transgenic line with muscle-specific overexpression of GrnA. In conclusion, we demonstrate a critical role for PGRN in the maintenance of MPC and suggest that muscle atrophy under PGRN loss may begin with MPC during postembryonic myogenesis.

Cloning and functional analysis of the proximal promoter region of the three GnRH genes from the silver sea bream (Sparus sarba)

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a major role in releasing pituitary gonadotropin and controlling vertebrate reproduction. In this study, three GnRH cDNAs, GnRH-I (sbGnRH; 348 bp), GnRH-II (cGnRH-II; 557 bp), and GnRH-III (sGnRH; 483 bp), were cloned from the brain of the silver sea bream (Sparus sarba). In order to understand how the expression of the GnRH isoforms was regulated in the brain, the promoter of each gene was cloned and analyzed. We found regulatory motifs in the promoters that were conserved in the GnRH promoters of tilapia and zebrafish, suggesting that these motifs play a critical role in GnRH regulation. We performed functional analyses and examined tissue-specific expression for each GnRH promoter using EGFP reporter fusions in zebrafish. The GnRH-I promoter was active in the forebrain area, including the olfactory bulb-terminal nerve area and peripheral preoptic areas; the GnRH-II promoter was active in the midbrain; and the GnRH-III promoter was active in the olfactory bulb. These results show that the GnRH promoters of the silver sea bream GnRH genes exhibit tissue-specific activity.