Transgenic expression of walleye dermal sarcoma virus rv-cyclin gene in zebrafish and its suppressive effect on liver tumor development after carcinogen treatment.

A retrovirus homologue gene of cellular cyclin D1, walleye dermal sarcoma virus rv-cyclin gene (orf A or rv-cyclin), was expressed in the livers of zebrafish under the control of liver fatty acid-binding protein (lfabp) promoter. To prevent possible fatality caused by overexpression of the oncogene, the GAL4/upstream activation sequence (GAL4/UAS) system was used to maintain the transgenic lines. Thus, both GAL4-activator [Tg(lfabp:GAL4)] and UAS-effector [Tg(UAS:rvcyclin)] lines were generated, and the rv-cyclin gene was activated in the liver after crossing these two lines. Since no obvious neoplasia phenotypes were observed in the double-transgenic line, cancer susceptibility of the transgenic fish expressing rv-cyclin was tested by carcinogen treatment. Unexpectedly, transgenic fish expressing rv-cyclin gene (rvcyclin+) were more resistant to the carcinogen than siblings not expressing this gene (rvcyclin-). Lower incidences of multiple and malignant liver tumors were observed in rvcyclin+ than in rvcyclin- fish, and the liver tumors in the rvcyclin+ group appeared later and were less malignant. These results suggest that expression of rv-cyclin protects the fish liver from carcinogen damage and delays onset of malignancy. These findings indicate that transgenic fish models are powerful systems for investigating mechanisms of inhibition and regression of liver tumors.

Development of a heat shock inducible gfp transgenic zebrafish line by using the zebrafish hsp27 promoter.

In the present study, a zebrafish hsp27 promoter was isolated and used to develop heat shock inducible gfp transgenic zebrafish. The endogenous hsp27 mRNAs were constitutively expressed from 4 hpf and increased in several regions of brain, heart and somites in early embryogenesis until 24 hpf. Subsequently, the expression was reduced significantly but maintained in the heart and ears. Heat shock induced hsp27 mRNAs in the blastoderm from 6 hpf and later in somites, branchial arches and several regions of brain. Similarly in hsp27-gfp transgenic zebrafish, constitutive GFP expression was observed from 11 hpf. GFP expression was mainly in the skin cells and increased to the peak level at 7 dpf, followed by a reduction. The constitutive GFP expression in the heart was initiated from 50 hpf and maintained even in the adult fish. After heat shock, GFP expression was mainly induced in the muscle in addition to a mild increase in the skin and heart. The early stages of the embryos were more sensitive than late stages as the time required for induced GFP expression in the muscle is shorter. Thus, the hsp27-gfp transgenic line generally recapitulates the expression pattern and heat shock inducibility of endogenous hsp27 RNAs. We also tested the potential of using the hsp27-gfp transgenic zebrafish embryos for heavy metal induction and demonstrated the inducibility of GFP expression by arsenic; this pattern of induction was also supported by examination of endogenous hsp27 mRNA.

Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression.

The zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype.

Transcriptome analysis of zebrafish embryogenesis using microarrays.

Zebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html).

A novel zebrafish kelchlike gene klhl and its human ortholog KLHL display conserved expression patterns in skeletal and cardiac muscles.

In this study, a novel gene, kelchlike (klhl) was identified in zebrafish by whole-mount in situ hybridization screen for important genes involved in embryogenesis. A full-length klhl cDNA was cloned and characterized. We found that klhl was a member of the kelch-repeat superfamily, containing two evolutionary conserved domains--broad-complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain, and kelch motif. Database mining revealed the presence of putative orthologs of klhl in human, mouse, rat, and pufferfish. klhl was determined to map to zebrafish linkage group (LG) 13 and was found to be syntenic with the proposed orthologs of klhl in human, mouse, and rat. In an effort to elucidate the function of klhl, klhl expression was investigated by Northern blot analysis and in situ hybridization. klhl is specifically expressed in the fast skeletal and cardiac muscle. Northern blot analyses show that the human ortholog, KLHL, is also specifically expressed in the skeletal muscles and heart. In silico analyses of rat expressed sequence tag (EST) clones corresponding to rat Klhl ortholog also indicate that its expression is also restricted to rat muscle tissues, suggesting a conserved role of klhl in vertebrates. The expression pattern of klhl, as well as the presence of the kelch repeats indicates a possible role for Klhl in the organization of striated muscle cytoarchitecture.