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 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.

Zebrafish eggs used as bioreactors for the production of bioactive tilapia insulin-like growth factors.

Multiple advantages-including the short generation time, large numbers of fertilized eggs, low cost of cultivation and easy maintenance favor the use of fish as bioreactors for the production of pharmaceutical proteins. In the present study, zebrafish eggs were used as bioreactors to produce mature tilapia insulin-like growth factors (IGFs) proteins using the oocyte-specific zona pellucida (zp3) promoter. The chimeric expression plasmids, pT2-ZP-tIGFs-IRES-hrGFP, in which hrGFP was used as reporter of tilapia IGFs expression, were designed to established Tg (ZP:tIGFs:hrGFP) transgenic lines for the expression of tilapia IGF-1 and IGF-2. Recombinant tilapia IGF-1 and IGF-2 were expressed as soluble forms in cytoplasm of fertilized eggs. The content level of tilapia IGF-1 and IGF-2 were 6.5 and 5.0% of the soluble protein, respectively. Using a simple Ni-NTA affinity chromatography purification process, 0.58 and 0.49 mg of purified tilapia IGF-1 and IGF-2 were obtained, respectively, from 650 fertilized eggs. The biological activity of the purified tilapia IGF-1 and IGF-2 was confirmed via a colorimetric bioassay to monitor the growth stimulation of zebrafish embryonic cells (ZF4), tilapia ovary cells (TO-2) and human osteosarcoma epithelial cells (U2OS). These results demonstrate that the use of zebrafish eggs as bioreactors is a promising approach for the production of biological recombinant proteins.

Thioacetamide accelerates steatohepatitis, cirrhosis and HCC by expressing HCV core protein in transgenic zebrafish Danio rerio.

Hepatocellular carcinoma (HCC) is one of the common cancers worldwide, caused by Hepatitis C virus (HCV) and hepatotoxins. Here we report the development of HCC in wild type as well as HCV core protein (HCP)-transgenic zebrafish upon treatment with a hepatotoxin, thioacetamide (TAA). Two-fold accelerated HCC development could be achieved in the TAA-treated transgenic fish, that is, the progression of the disease in TAA-treated wild type zebrafish developed HCC in 12 weeks whereas that of HCP-transgenic zebrafish shortened the HCC progression to 6 weeks. Histopathological observation showed the specific pathological features of HCC. The HCC progression was confirmed through RT-PCR that revealed an up and down regulation of different marker genes at various stages of HCC progression such as, steatohepatitis, fibrosis and HCC. Moreover, HCV core protein expressed in the HCP-transgenic zebrafish and TAA synergistically accelerate the HCC development. It must be mentioned that, this is the first report revealing HCV core protein along with TAA to induce HCC in zebrafish, particularly, in a short period of time comparing to mice model. As zebrafish has already been considered as a good human disease model and in this context, this HCC-zebrafish model may serve as a powerful preclinical platform to study the molecular events in hepatocarcinogenesis, therapeutic strategies and for evaluating chemoprevention strategies in HCC.

Two distinct teleost hepatocyte nuclear factor 1 genes, hnf1alpha/tcf1 and hnf1beta/tcf2, abundantly expressed in liver, pancreas, gut and kidney of zebrafish.

Two distinct forms of zebrafish hepatocyte nuclear factor 1 (hnf1) were identified and referred to as hnf1alpha/tcf1 and hnf1beta/tcf2. Both hnf1 genes were shown to be expressed abundantly in liver, pancreas, gut and kidney. Zebrafish HNF1alpha and HNF1beta proteins contain all HNF1 signature domains including the dimerization domain, POU-like domain and atypical homeodomain. Sequence and phylogenetic analysis reveals that zebrafish hnf1alpha is closer to tetrapodian hnf1alpha than to tetrapodian hnf1beta and zebrafish hnf1beta is highly conserved with tetrapodian hnf1beta. Existences of hnf1alpha and hnf1beta in teleost zebrafish, tilapia and fugu suggest that hnf1 gene duplication might occur before the divergence of teleost and tetrapod ancestors. Zebrafish hnf1alpha and hnf1beta genes were mapped to linkage group LG8 and LG15 in T51 panel by RH mapping and are composed of 10 and 9 exons, respectively. Zebrafish hnf1beta gene with at least 11 genes in LG15 was identified to maintain the conserved synteny with those of human in chromosome 17 and those of mouse in chromosome 11. Our results indicate that distinct hnf1alpha and hnf1beta genes in teleosts had been evolved from the hnf1 ancestor gene of chordate.

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.

Stage-specific expression of TNFα regulates bad/bid-mediated apoptosis and RIP1/ROS-mediated secondary necrosis in Birnavirus-infected fish cells.

Infectious pancreatic necrosis virus (IPNV) can induce Bad-mediated apoptosis followed by secondary necrosis in fish cells, but it is not known how these two types of cell death are regulated by IPNV. We found that IPNV infection can regulate Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic death pathways via the up-regulation of TNFα in zebrafish ZF4 cells. Using a DNA microarray and quantitative RT-PCR analyses, two major subsets of differentially expressed genes were characterized, including the innate immune response gene TNFα and the pro-apoptotic genes Bad and Bid. In the early replication stage (0-6 h post-infection, or p.i.), we observed that the pro-inflammatory cytokine TNFα underwent a rapid six-fold induction. Then, during the early-middle replication stages (6-12 h p.i.), TNFα level was eight-fold induction and the pro-apoptotic Bcl-2 family members Bad and Bid were up-regulated. Furthermore, specific inhibitors of TNFα expression (AG-126 or TNFα-specific siRNA) were used to block apoptotic and necrotic death signaling during the early or early-middle stages of IPNV infection. Inhibition of TNFα expression dramatically reduced the Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic cell death pathways and rescued host cell viability. Moreover, we used Rip1-specific inhibitors (Nec-1 and Rip1-specific siRNA) to block Rip1 expression. The Rip1/ROS-mediated secondary necrotic pathway appeared to be reduced in IPNV-infected fish cells during the middle-late stage of infection (12-18 h p.i.). Taken together, our results indicate that IPNV triggers two death pathways via up-stream induction of the pro-inflammatory cytokine TNFα, and these results may provide new insights into the pathogenesis of RNA viruses.

Nitroreductase-mediated gonadal dysgenesis for infertility control of genetically modified zebrafish.

Genetically modified (GM) fish with desirable features such as rapid growth, disease resistance, and cold tolerance, among other traits, have been established in aquaculture. However, commercially available GM fish are restricted because of global concerns over the incomplete assessments of food safety and ecological impact. The ecological impact concerns include gene flow and escape of the GM fish, which may cause extinction of wild natural fish stocks. Infertility control is a core technology for overcoming this obstacle. Although polyploidy technology, GnRH-specific antisense RNA, and RNAi against GnRH gene expression have been used to cause infertility in fish, these approaches are not 100% reliable and are not heritable. In the present study, zebrafish was used as a model to establish an inducible platform of infertility control in GM fish. Nitroreductase, which converts metronidazole substrate into cytotoxin, was fused with EGFP and expressed specifically by oocytes in the Tg(ZP:NTR-EGFP) by a zona pellucida promoter. Through consecutive immersion of metronidazole from 28 to 42 days posthatching, oocyte-specific EGFP expression was eliminated, and atrophy of the gonads was detected by anatomical analysis. These findings reveal that oocyte-specific nitroreductase-mediated catalysis of metronidazole blocks oogenesis and leads to an undeveloped oocyte. Furthermore, oocyte cell death via apoptosis was detected by a TUNEL assay. We found that the gonadal dysgenesis induced by metronidazole resulted in activation of the ovarian killer gene bok, which is a proapoptotic gene member of the Bcl-2 family and led to infertility. These results show that oocyte-specific nitroreductase-mediated catalysis of metronidazole can cause reliable infertility in zebrafish and could potentially be used as a model for other aquaculture fish species.

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.

XBP-1, a key regulator of unfolded protein response, activates transcription of IGF1 and Akt phosphorylation in zebrafish embryonic cell line.

The unfolded protein response (UPR) is a conserved and adaptive cellular response to increase cell survival during ER stress. XBP-1 spliced form (XBP-1S) generated by IRE1 endoribonuclease is a key transcriptional regulator in UPR to activate genes involved in protein folding and degradation to restore ER function. Although Akt activation was suggested to be a pro-survival pathway activated during ER stress, the signal to trigger Akt is still not clear. In this study, we report IGF1 transcription and Akt phosphorylation are enhanced in XBP-1S stably overexpressed clone of zebrafish embryonic cell line (ZF4). In addition, zebrafish IGF1 intron1 with predicted UPRE (XBP-1S binding sites) and ERSE (ATF6/XBP-1S binding site) linked with basal promoter could be activated by XBP-1S, not by XBP-1 unspliced form (XBP-1U). Furthermore, we demonstrate that expression of endogenous IGF1 is transiently induced as XBP-1 splicing during ER stress in parallel to ER chaperone GRP78/Hspa5 and ER resided E3 ubiquitin ligase Synoviolin in ZF4 cells by quantitative PCR. Our results suggest zebrafish XBP-1S not only activates genes responsible for protein folding, transporting, glycosylation and ER associated degradation but also activates anti-apoptosis signal via IGF1/Akt pathway in unfolded protein response to cope with ER stress.

Co-induction of hepatic IGF-I and progranulin mRNA by growth hormone in tilapia, Oreochromis mossambiccus.

Like IGF-I, progranulin (pgrn) is a growth factor involved in tumorigenesis and wound healing. We report here the identification and characterization of pgrn cDNA in tilapia and the regulation of its expression by growth hormone (GH). The tilapia pgrn cDNA was cloned by RT-PCR amplification, using gene specific oligonucleotides as amplification primers. The cDNA contains an open reading frame encoding a peptide of 206 amino acid residues (aa) that contains a presumptive signal peptide (23 aa) and two repeat units of granulin (grn, 51 and 52 aa, respectively) franked by a GAP of 49 aa and the carboxyl terminus with 31 aa. The two predicted grn peptides are arranged in tandem repeats interrupted by a GAP peptide. RT-PCR analysis revealed that high levels of prgn mRNA were present in several tissues such as spleen, gastric cecum, intestine, fat tissue, gill, kidney, eye and pancreas, and lower levels in liver, muscle, heart, brain, skin and stomach. Administration of a single dose (500 ng/g body weight) of recombinant seabream growth hormone (rbGH) by intraperitoneal (ip) injection into one-month-old tilapia resulted in an obvious increase of IGF-I and pgrn mRNA (2.7-fold and 2.5-fold, respectively) in the liver at three hours post-GH treatment. The peptide levels of pgrn in the liver of GH-treated fish also were substantially induced over controls at 12h post-GH treatment as detected by western immuno-blot analysis. The co-induction of IGF-I and pgrn following GH treatment may suggest the involvement of pgrn in GH regulated growth in tilapia.

Phosphatidylserine receptor is required for the engulfment of dead apoptotic cells and for normal embryonic development in zebrafish.

During development, the role of the phosphatidylserine receptor (PSR) in the removal of apoptotic cells that have died is poorly understood. We have investigated this role of PSR in developing zebrafish. Programmed cell death began during the shield stage, with dead cells being engulfed by a neighboring cell that showed a normal-looking nucleus and the nuclear condensation multi-micronuclei of an apoptotic cell. The zebrafish PSR engulfing receptor was cloned (zfpsr), and its nucleotide sequence was compared with corresponding sequences in Drosophila melanogaster (76% identity), human (74%), mouse (72%) and Caenorhabditis elegans (60%). The PSR receptor contained a jmjC domain (residues 143-206) that is a member of the cupin metalloenzyme superfamily, but in this case serves an as yet unknown function(s). psr knockdown by a PSR morpholino oligonucleotide led to accumulation of a large number of dead apoptotic cells in whole early embryo. These cells interfered with embryonic cell migration. In addition, normal development of the somite, brain, heart and notochord was sequentially disrupted up to 24 hours post-fertilization. Development could be rescued in defective embryos by injecting psr mRNA. These results are consistent with a PSR-dependent system in zebrafish embryos that engulfs apoptotic cells mediated by PSR-phagocytes during development, with the system assuming an important role in the normal development of tissues such as the brain, heart, notochord and somite.