Molecular cloning and functional analysis of the zebrafish luteinizing hormone beta subunit (LH) promoter.

The luteinizing hormone (LH) plays important roles in vertebrate reproduction. In the present study, we cloned and characterized the zebrafish (Danio rerio) LH subunit gene structure and promoter region. Analysis of 3.0 kb (LH3.4K~5'UTR) of the LH subunit proximal promoter region displayed maximal promoter activity in a tilapia ovary cell line (TO2 cells) after treatment with gonadotropin-releasing hormone (GnRH). Transient expression experiments with a 5'-deletion revealed at least 10 regulatory regions in the zebrafish LH subunit gene. Compared to the molecular mechanisms of other vertebrates, GnRH treatment led to the activation of zebrafish LH subunit gene transcription in ovary cells. We demonstrated that LH subunit gene transcription increased with 6 h of treatment with GnRH but was repressed by protein kinase C, mitogen-activated protein kinase, and calcium in the TO2 cell line. To study promoter-specific expression, we constructed an LH subunit (LH3.4k~5'UTR) promoter region-driven green fluorescent protein (GFP), and the results indicated that LH promoter-driven GFP transcripts appeared in the pituitary gland. For the gene knockdown study, we targeted knockdown of the LH subunit gene by two antisense morpholino oligonucleotides that resulted in serious abnormalities and death during zebrafish embryogenesis. These results suggest that the LH plays important roles in reproduction and general embryonic development in zebrafish.

Molecular cloning and functional analysis of the zebrafish follicle-stimulating hormone (FSH)beta promoter.

In the present study, we cloned and characterized a zebrafish follicle-stimulating hormone (zfFSH)beta promoter with deletion fragments transfected into a tilapia ovary (TO2) cell line, and demonstrated that the zfFSHbeta promoter responded to 6h of gonadotropin-releasing hormone (GnRH) treatment by activating calcium influx and protein kinase C (PKC), but after 24h, GnRH induction was generated by activation of extracellular-regulated kinase (ERK)1/2 and repression by PKC. Furthermore, to study the promoter-specific expression, we constructed a series of FSHbeta (4.0-, 3.0-, 2.0-, and 1.0-kb) promoter-driven green fluorescent protein (GFP) fragments encoding the GFP complementary DNA transgene which was microinjected into zebrafish embryos. Morphological studies of transgenic zebrafish indicated that the FSHbeta promoter-driven GFP transcripts appeared in the heart, skin, and vertebrae.

Stable expression in a Chinese hamster ovary (CHO) cell line of bioactive recombinant chelonianin, which plays an important role in protecting fish against pathogenic infection.

Chelonianin, originally isolated from the shrimp (Penaeus monodon), exhibits antimicrobial effects in vitro and in vivo and is used to treat infectious fish diseases. Herein, we report that the recombinant chelonianin protein fused to a fluorescent protein (rcf protein) was expressed from a stably transfected Chinese hamster ovary (CHO) cells. The in vitro experiments showed that the rcf protein exhibited antimicrobial activity against several bacteria, while the recombinant fluorescent protein alone did not. In addition, pretreatment and post-treatment with the rcf protein were both effective in promoting a significant decrease in fish mortality and decreasing the number of infectious bacteria. We utilized the quantitative reverse-transcriptase polymerase chain reaction technique to survey the levels of gene expressions of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide synthase 1 induced in response to bacterial infection in experiments with tilapia (Oreochromis mossambicus). Our results indicated that the rescue of fish treated with the rcf protein may involve regulation of TNF-alpha expression. Collectively, chelonianin inhibited the production of an inflammatory mediator and reduced mortality in fish during bacterial challenge, suggesting that it has potential as a therapeutic or prophylactic drug for use against bacterial infectious diseases.

Application of RNAi technology to the inhibition of zebrafish GtHalpha, FSHbeta, and LHbeta expression and to functional analyses.

Zebrafish (Danio rerio) were used as a model fish, and the technique of RNA interference (RNAi) was employed to knockdown three subunits of the gonadotropin alpha (GtHalpha, common alpha), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) genes. Three short-hairpin RNA (shRNA) expression vectors and three mismatched shRNA expression vectors as controls for each subunit gene were constructed, and the depression efficiency was tested in vivo by microinjection; the RNA or protein expression levels of the GtH genes were monitored by RT-PCR, Southern blotting, and green fluorescent protein (GFP) analyses. Expression of GtH mRNA was obviously and more efficiently depressed by GtHalpha RNAi expression compared with the other two subunits. A GtHalpha morpholino analysis showed that the GtHalpha morpholino led to suppression of embryonic development and the production of embryonic mutants as a result of an injection of GtHalpha -shRNA. Taken together, these results show that GtHalpha-shRNA, which more efficiently targets RNAi, may have an essential role in the further development of sterility technology of transgenic fish for biosafety purposes.

Functional analysis of mitogen-activated protein kinase-3 (MAPK3) and its regulation of the promoter region in zebrafish.

Mitogen-activated protein kinase (MAPK) plays a pivotal role in intracellular actions in response to a variety of extracellular stimuli. Real-time reverse-transcription polymerase chain reaction analysis of MAPK3 tissue distribution in zebrafish showed significant differences in the fin and liver compared with muscle. A 1.2-kilobase (kb) pair and a 2.3-kb fragment of the 5'-flanking region displayed minimal promoter activity in the zebrafish liver (ZFL) and HeLa cell lines after treatment with insulin-like growth factors (IGF-I and IGF-II). Targeted knockdown of the MAPK3 gene by two antisense morpholino oligonucleotides revealed that although the zebrafish MAPK3 MO 1-targeted sequence was located at 5' untranslated region and the zebrafish MAPK3 MO 2-targeted sequence was located in the mature peptide region, similar results were shown in zebrafish for disruption of notochord development, with the whole body exhibiting distortion. From a comparative point of view, this study of the MAPK3 gene in zebrafish might not correlate well with previously published studies on mice. These molecular results suggest that MAPK3 plays an important role in whole-body development and is required for general embryonic development. Finally, MAPK3 may play important roles in fish cell growth.

Cloning and expression analysis of a protein kinase C gene, PKCmu, and its regulation of the promoter region in zebrafish.

The cDNA and genomic DNA of zebrafish (Danio rerio) protein kinase Cmu (PKCmu), with its promoter region, were obtained. The 508-amino acid zebrafish PKCmu has 86.17% similarity to human PKCmu. Real-time reverse-transcription polymerase chain reaction analysis with starvation and hormonal treatment found significant differences between the control group and the experimental group after 14 days of starvation. After injecting insulin-like growth factor II (IGF-II), growth hormone (GH), insulin, or human chorionic gonadotropin, significant differences were observed between the control and experimental groups 24 h after treatment. After injecting the gonadotropin-releasing hormone or luteotropin-releasing hormone, significant differences were seen between the control and experimental groups 15 h after treatment. These results suggest that in vivo PKCmu expression is regulated by the insulin family or by the GH, but other sex hormones produced a significant expression level more quickly than the insulin family and GH. The zebrafish PKCmu gene is located on zebrafish chromosome 17 and consists of 16 exons. A 2.6 kilobase pair on the 5' flanking region displayed maximal promoter activity in the zebrafish liver (ZFL) cell line after treatment with IGF-I, IGF-II, and GH. However, a 1.6 kilobase pair on the 5' flanking region displayed maximal promoter activity in the HeLa cell line after treatment with IGF-I, IGF-II, and GH. Finally, PKCmu may have important nuclear effects on cell growth and may involve nuclear localization. By transiently transfecting ZFL cells with various zebrafish PKCmu segments, we identified a nuclear localization signal: the amino acid sequence between amino acids 206 and 209 was able to predominantly direct enhanced green fluorescence protein (EGFP) into the nucleus, whereas a deletion of this motif abrogated the nuclear localization property.

The physiological role of CTGF/CCN2 in zebrafish notochond development and biological analysis of the proximal promoter region.

During mouse embryogenesis, CTGF/CCN2 is expressed in zones containing hypertrophic chondroctyes and calcifying cartilage such as long bones, ribs, vertebral column, and phalanges. But in fish, its expression is yet unclear. Development of the vertebrae is morphologically similar among vertebrates, indicating that the underlying mechanism regulating the process is highly conserved during evolution. Analysis of 3.2kb of the CTGF/CCN2 proximal promoter sequence revealed a consensus TATAA box, putative AP1, Brn-2, CdxA, C/EBP alpha, C/EBP beta, C-Ets-, delta E, HFH-2, and HSF2 binding sites. Transient expression experiments with a 5'-deletion revealed at least 4 regulatory regions in the zebrafish CTGF/CCN2 gene, 2 with a stimulatory effect on transcription and 2 with an apparent inhibitory effect after IGF-I treatment in the ZFL cell line. To study the promoter-specific expression, we constructed a series of CTGF/CCN2 (3.0-, 2.5-, 2.0-, 1.5-, 1.0-, and 0.4-kb) promoter-driven green fluorescent protein (GFP) fragments encoding the GFP cDNA transgene which was microinjected into zebrafish embryos. Morphological studies of transgenic zebrafish indicated that the CTGF/CCN2 promoter-driven GFP transcripts appeared in the notochord. Targeted knockdown of the CTGF/CCN2 gene by two antisense morpholino oligonucleotides resulted in disruptions to notochord development. From a comparative point of view, this study of the CTGF/CCN2 gene in zebrafish may correlate well with those previously published on the mouse. These molecular results suggest that CTGF/CCN2 plays an important role in notochord development and is required for general embryonic development.