Association Between Muscle Growth and Transcription of a Mutant MSTN Gene in Olive Flounder (Paralichthys olivaceus).

Myostatin (MSTN, also known as growth differentiation factor-8 (GDF-8)), a member of the transforming growth factor ß (TGF-ß) superfamily, functions as a negative regulator of skeletal muscle development and growth. However, it is also expressed in a wide range of tissues in fish and thus may have more diverse roles in this group than in mammals. In this study, we assessed the genome-wide transcriptional expression pattern associated with the CRISPR/Cas9-mutated MSTN gene in the olive flounder (Paralichthys olivaceus) in association with changes in cell proliferation and transportation processes. There were no differences in the hepatosomatic index, and the growth of male and female fish increased in the F1 progeny of the MSTN mutants. Furthermore, the histopathological analysis showed that myostatin editing resulted in a 41.24% increase in back muscle growth and 46.92% increase in belly muscle growth in male flounder compared with normal flounder, and a 16.01% increase in back muscle growth and 14.26% increase in belly muscle growth in female flounder compared with normal flounder. This study demonstrates that editing of the myostatin gene enhances muscle growth in olive flounder, with a notably more pronounced effect observed in males. Consequently, myostatin-edited male flounder could represent a valuable asset for the flounder aquaculture industry.

Analysis of Polymeric Immunoglobulin Receptor Expression in Olive Flounder (Paralichthys olivaceus) against Viral Hemorrhagic Septicemia Virus.

Polymeric immunoglobulin receptor (pIgR) mediates the transfer of polymeric immunoglobulin to protect organisms and is one of the most important mucosal effectors. In this study, the developmental stage- and tissue-specific expression of pIgR were observed before virus inoculation in olive flounder. pIgR was gradually expressed until the formation of immune tissue, exhibiting high expression in the late juvenile period; thereafter, pIgR expression gradually decreased and exhibited high expression in the spleen and skin. Moreover, pIgR expression after viral hemorrhagic septicemia virus infection was high in the kidney and spleen tissues at high density and low at low density. The results of this study can provide a basis for future studies on breeding density, virus expression, and immune system studies in fish.

Zebrafish Klf11b is Required to Maintain Cell Viability by Inhibiting p53-Mediated Apoptosis.

Krüppel-like factor 10 (KLF10) regulates various cellular functions, such as proliferation, differentiation and apoptosis, as well as the homeostasis of several types of tissue. In the present study, we attempted a loss-of-function analysis of zebrafish Klf11a and Klf11b, which constitute human KLF10 homologs. Embryos injected with klf11b-morpholino (MO) showed developmental retardation and cell death, whereas klf11a-MO-injected embryos showed normal development. In klf11b-MO-injected embryos, a dramatic increase in the amount of zebrafish p53 mRNA might be the cause of the increase in that of bax. The degree of apoptosis decreased in the klf11b-MO and p53-MO co-injected embryos. These findings imply that KLF10 is a negative regulator of p53-dependent transcription, suggesting that the KLF10/p53 complex may play an important role in apoptosis for maintenance of tissue homeostasis during embryonic development.

Characterization of TRIM16, a member of the fish-specific finTRIM family, in olive flounder Paralichthys olivaceus.

Tripartite motif-containing (TRIM) proteins are conserved throughout the metazoan kingdom, and the TRIM subset finTRIM is highly diversified in fish. We isolated TRIM16 cDNA, a member of the finTRIM family, from the olive flounder Paralichthys olivaceus (PoTRIM16). PoTRIM16 contained a 1,725-bp coding sequence encoding a 574-amino acid polypeptide, which in turn contained a really interesting new gene (RING) finger domain, B-box-type zinc finger (B-BOX), nuclease SbcCD subunit C (SbcC), structural maintenance of chromosome (SMC prok B), and stonustoxin (SNTX) subunit alpha (SPRY-PRY-SNTX). Multiple alignment of related sequences revealed that PoTRIM16 showed 86.63-97.40% identity with fish orthologues, and a phylogenetic tree was constructed of vertebrates. PoTRIM16 mRNA was detected in all tissues examined; levels were highest in the eye and ovary. PoTRIM16 mRNA expression was investigated during early development. Under VHSV infection, PoTRIM16 mRNA was downregulated in the liver of P. olivaceus. This is the first study to characterize fish-specific finTRIM in P. olivaceus, which may play a role in the immune response against virus infection.

A single amino acid variation of NV protein of viral hemorrhagic septicemia virus increases protein stability and decreases immune gene expression.

Viral hemorrhagic septicemia virus (VHSV) causes severe mortality among more than 90 fish species. The 11 kb viral genome encodes six proteins including nonvirion protein (NV). In previous study, we reported that NV gene variations of VHSV decrease cellular energy metabolism. Among several NV mutant proteins, NV-S56L showed the highest cellular energy deprivation. Based on this finding, we further examined a molecular mechanism of one amino acid (S56L) change on differential cellular dysregulation. In the fish cells, the NV-S56L protein showed an increased level of cellular expression than normal and other mutant NV proteins without change of mRNA expression. Using cycloheximide treatment for exclude de novo NV protein expression, NV-S56L had an extensive half-life of intracellular protein. The proteasome inhibitor, MG-132, treatment recovered the all NV protein levels. The ubiquitination of NV was increased in the treatment of MG132 via inhibition of the ubiquitin/proteasome system process. Finally, increased protein stability of NV-S56L led to downregulation of NF-κB response immune gene expression. These results indicate that the prolonged protein stabilization of NV protein variant (NV-S56L) increases its pathological duration and might eventually lead to high virulence activity in the host fish cell.

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells.

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

Analysis of Tissue-Specific Interferon Regulatory Factor 3 (IRF3) Gene Expression against Viral Infection in Paralichthys olivaceus.

Interferon Regulatory Factor 3 (IRF3) is a member of interferon-regulated transcription factor family and is known to play an important role in the innate immune response against viral infections. In this study, the expression of IRF3 in different tissues, developmental stages, and stocking densities of olive flounder was investigated. The expression of IRF3 was observed to gradually increase in early-stage juvenile fish. The highest expression was observed in later-stage juvenile fish when immune tissues were formed. High IRF3 expression was observed in the muscles and the brain tissues. The expression of IRF3 was observed in fish at different stocking densities after viral hemorrhagic septicemia virus (VHSV) infection. It yielded an interesting expression pattern in the muscles and the brain tissues of fish stocked at low density. These observations can be used as basic data for the study of the expression of immune response-related genes against viruses based on stocking density and immune systems in other fish species.

Expression Analysis of the Caspase10 from Olive Flounder (Paralichthys olivaceus) against Viral Hemorrhagic Septicemia Virus (VHSV) Challenge.

The caspase10 encodes an initiating caspase that plays an important role in the maintaining the cellular homeostasis by regulating the steps involved in the immune response and cell death. We investigated the expression of caspase10 during the different developmental stages and in olive flounder tissues. Caspase10 increased in the late stage of the formation of immune tissue, and high expression was observed in the gills, kidney, skin, and spleen. The current study analyzed the expressional changes of caspase10 in olive flounder infected with viral hemorrhagic septicemia virus (VHSV). One of the major causes of mass mortality, VHSV infection in olive flounder attributes to significant expression of caspase10 in the gills, spleen, skin, and kidneys. The results indicate a close association of caspase10 expression with the immune response to VHSV infection in olive flounder. The observations could form the basis data for exploration of other fish immune system.

First report of cathepsin E in a teleost (Korean rose bitterling, Rhodeus uyekii): Molecular characterisation and tissue distribution.

We isolated and characterised a cDNA encoding the aspartic protease cathepsin E (CTSE) in Korean rose bitterling, Rhodeus uyekii. The full-length Rhodeus uyekii CTSE (RuCTSE) cDNA (1396 bp) contains an open reading frame of 1218 bp, encoding 405 amino acids. Alignment of multiple CTSE protein sequences revealed that two of the aspartyl protease active site residues and a disulphide bond were well-conserved among the other CTSE sequences. Phylogenetic analysis revealed that RuCTSE is most closely related to freshwater fish cathepsin E. RuCTSE is widely expressed in the liver, spleen, ovary, testis, brain, eye, intestine, muscle, fin, stomach, and kidney. This first report of teleost CTSE will provide important information related to the identification of other cathepsin E genes in various fish species and will serve as a useful molecular tool to help clarify biological activities in other teleosts.

The Regulatory Region of Muscle-Specific Alpha Actin 1 Drives Fluorescent Protein Expression in Olive Flounder Paralichthys olivaceus.

To develop a promoter capable of driving transgene expression in non-model fish, we identified and characterized the muscle-specific alpha-actin gene in olive flounder, Paralichthys olivaceus (PoACTC1). The regulatory region of PoACTC1 includes putative regulatory elements such as a TATA box, two MyoD binding sites, three CArG boxes, and a CCAAT box. Microinjection experiments demonstrated that the regulatory region of PoACTC1, covering from -2,126 bp to +751 bp, just prior to the start codon, drove the expression of red fluorescent protein in developing zebrafish embryos and hatching olive flounder. These results suggest that the regulatory region of PoACTC1 may be useful in developing a promoter for biotechnological applications such as transgene expression in olive flounder.

Generation of motor neurons requires spatiotemporal coordination between retinoic acid and Mib-mediated Notch signaling.

Mind bomb (Mib) is an E3 ubiquitin ligase that activates the Notch signaling pathway. A previous study demonstrated that the generation of late-born GABAergic neurons may be regulated by the interplay between Mib and retinoic acid (RA). However, the relationship between Mib function and the retinoid pathway during the generation of late-born motor neurons remains unclear. We investigated the differentiation of neural progenitors into motor neurons by inhibition of Notch signaling and administration of RA to Tg[hsp70-Mib:EGFP] embryos. The number of motor neurons in the ventral spinal cord increased or decreased depending on the temporal inhibition of Mib-mediated Notch signaling. Inhibition of the retinoid pathway by citral treatment had a synergistic effect with overexpression of Mib:EGFP on the generation of ectopic motor neurons. Additionally, the proteolytic fragment of Mib was detected in differentiated P19 cells following treatment with RA. Our observations imply that the function of Mib may be attenuated by the retinoid pathway, and that Mib-mediated Notch signaling and the retinoid pathway play critical roles in the spatiotemporal differentiation of motor neurons.

Development and Characterization of a New Cell Line from Olive Flounder Paralichthys olivaceus.

A new embryonic cell line (OFEC-17FEN) derived from olive flounder Paralichthys olivaceus was developed. OFEC-17FEN cells were subcultured for <30 passages over ~200 days. OFEC-17FEN cells had a doubling time of 114.34 h and modal diploid chromosome number was 48. The pluripotency genes POU5f1 and NANOG were expressed in OFEC-17FEN cells. However, the lack of several pluripotency-related genes expression indicates that OFEC-17FEN cells are not stem cells. OFEC-17FEN cells transfected with plasmid pEGFP-c1 exhibited a strong green fluorescent signal at 48 h after transfection. Accordingly, OFEC-17FEN cells may be useful for both basic research and biotechnological application.

Molecular characterization of Rhodeus uyekii tripartite motif protein 1 (TRIM1) involved in IFN-γ/LPS-induced NF-κB signaling.

The tripartite motif-containing (TRIM) proteins are involved in a wide range of cellular processes, and the role of TRIM1 in immunity has been explored. However, fundamental studies on fish TRIM1 are lacking. In this study, we cloned and characterized TRIM1 cDNA from the Korean rose bitterling, Rhodeus uyekii (RuTRIM1). Two RuTRIM1 isoforms (RuTRIM1-X1 and RuTRIM1-X2) were identified. The coding sequence (CDS) of RuTRIM1-X1 comprised 2157 bp encoding a 718-aa protein, and the CDS of RuTRIM1-X2 comprised 1929 bp encoding a 642-aa protein. Both RuTRIM1 isoforms contained a RING finger domain, B-box 1, B-box 2, coiled-coil domain, COS box, FN3 motif, and PRY/SPRY domain. The deduced RuTRIM1-X1 and RuTRIM1-X2 proteins had high amino acid identity (76.27-98.89%) with orthologs from various other species, and a phylogenetic tree was constructed. RuTRIM1-X1 and RuTRIM1-X2 mRNA were expressed in all tissues examined, with the highest expression levels detected in the hepatopancreas. During early development, RuTRIM1-X1 and RuTRIM1-X2 mRNA levels changed differently from the gastrula period to the first feeding stage. An in vivo ubiquitination assay showed that RuTRIM1 exhibited RING-dependent E3 ubiquitin ligase activity, mainly by comparing RuTRIM1-X2 to RuTRIM1-X1. The subcellular localization of the two RuTRIM1 protein isoforms was characterized, revealing that they formed aggregates in cytoplasmic bodies in Raw264.7 cells. Interferon-γ/lipopolysaccharide-induced nuclear factor-κB signaling was negatively regulated by RuTRIM1-X1 and RuTRIM1-X2, and the negative effect was reversed in RING deletion mutants. To our knowledge, this is the first study to characterize fish TRIM1, which may play a role in the inflammatory response.

Microarray analysis of gene expression in olive flounder liver infected with viral haemorrhagic septicaemia virus (VHSV).

The most fatal viral pathogen in olive flounder Paralichthys olivaceus, is viral hemorrhagic septicemia virus, which afflicts over 48 species of freshwater and marine fish. Here, we performed gene expression profiling on transcripts isolated from VHSV-infected olive flounder livers using a 13 K cDNA microarray chip. A total of 1832 and 1647 genes were upregulated and down-regulated over two-fold, respectively, after infection. A variety of immune-related genes showing significant changes in gene expression were identified in upregulated genes through gene ontology annotation. These genes were grouped into categories such as antibacterial peptide, antigen-recognition and adhesion molecules, apoptosis, cytokine-related pathway, immune system, stress response, and transcription factor and regulatory factors. To verify the cDNA microarray data, we performed quantitative real-time PCR, and the results were similar to the microarray data. In conclusion, these results may be useful for the identification of specific genes or for the diagnosis of VHSV infection in flounder.

Molecular and Functional Characterization of Thioredoxin 1 from Korean Rose Bitterling (Rhodeus uyekii).

Thioredoxin is a multifunctional antioxidant enzyme that belongs to the reductase family. In this study, we cloned and characterized thioredoxin 1 cDNA from the Korean rose bitterling Rhodeus uyekii (RuTrx). The full-length RuTrx cDNA consists of 674 bp with a 324 nt open reading frame (ORF) encoding a 107 aa protein. The deduced RuTrx amino acid sequence indicated a characteristic redox active site, (31)WCGPC(35). Pairwise alignment revealed RuTrx amino acid identity (55.1%-83.2%) with orthologs from various species of mammalia, amphibia, fish and bird. Phylogenetic analysis was conducted to determine the evolutionary position of RuTrx. Expression analysis showed that RuTrx transcripts were present in all of the tissues examined, and was high in the hepatopancreas of R. uyekii. During early development, the expression of RuTrx transcripts was increased. Recombinant RuTrx protein (rRuTrx) was tested for its capacity to serve as an antioxidant enzyme using a metal-catalyzed oxidation (MCO) system. The ability of rRuTrx to protect against supercoiled DNA cleavage due to oxidative nicking increased in a dose-dependent manner. In Raw264.7 cells, Dihydroethidium (DHE) staining for ROS production indicated the antioxidant activity of rRuTrx. Together, these findings suggest that RuTrx may play a role in maintaining the redox state balance in Korean rose bitterling R. uyekii.

RNA-Seq-based transcriptome analysis of Korean rose bitterling (Rhodeus uyekii) exposed to synthetic estrogen 17-α-ethinylestradiol (EE2).

The potential impact of natural and synthetic estrogens on aquatic ecosystems has become a subject of great interest in recent years. One synthetic estrogen, 17-alpha-ethinylestradiol (EE2), is present in municipal sewage discharges and causes gonad alterations in various fish species. To understand the possible damage caused by EE2, male Rhodeus uyekii were exposed to 100 ng/L EE2 for 7 days. RNA-Seq was performed to assess the effects of EE2 on gene expression in hepatic and skin tissues. The analysis revealed that EE2 induced the expression of various genes, including sex hormone genes, anti-Mullerian hormone, vitellogenin, and estrogen receptor alpha; cancer genes, breast cancer anti-estrogen resistance protein 3, caveolin 2, and Smad2; and apoptotic genes, p53, Bcl-2, TNF-α, and WDR36. These results suggest that the synthetic estrogen EE2 disturbs the endocrine system and regulates both carcinogenic and apoptotic gene expressions in R. uyekii.

Four Members of Heat Shock Protein 70 Family in Korean Rose Bitterling (Rhodeus uyekii).

Heat shock protein (HSP) 70, the highly conserved stress protein families, plays important roles in protecting cells against heat and other stresses in most animal species. In the present study, we identified and characterized four Hsp70 (RuHSP4, RuHSC70, RuHSP12A, RuGRP78) family proteins based on the expressed sequence tag (EST) analysis of the Korean rose bitterling R. uyekii cDNA library. The deduced RuHSP70 family has high amino acid identities of 72-99% with those of other species. Phylogenetic analysis revealed that RuHsp70 family clustered with fish groups (HSP4, HSC70, HSP12A, GRP78) proteins. Quantitative RT-PCR analysis showed the specific expression patterns of RuHsp70 family members in the early developmental stages and several tissues in Korean rose bitterling. The expression of 4 groups of Hsp70 family was detected in all tested tissue. Particularly, Hsp70 family of Korean rose bitterling is highly expressed in hepatopancreas and sexual gonad (testis and ovary). The expression of Hsp70 family was differentially regulated in accordance with early development stage of Rhodeus uyekii.