Molecular conservation of estrogen-response associated with cell cycle regulation, hormonal carcinogenesis and cancer in zebrafish and human cancer cell lines.

BACKGROUND: The zebrafish is recognized as a versatile cancer and drug screening model. However, it is not known whether the estrogen-responsive genes and signaling pathways that are involved in estrogen-dependent carcinogenesis and human cancer are operating in zebrafish. In order to determine the potential of zebrafish model for estrogen-related cancer research, we investigated the molecular conservation of estrogen responses operating in both zebrafish and human cancer cell lines. METHODS: Microarray experiment was performed on zebrafish exposed to estrogen (17ß-estradiol; a classified carcinogen) and an anti-estrogen (ICI 182,780). Zebrafish estrogen-responsive genes sensitive to both estrogen and anti-estrogen were identified and validated using real-time PCR. Human homolog mapping and knowledge-based data mining were performed on zebrafish estrogen responsive genes followed by estrogen receptor binding site analysis and comparative transcriptome analysis with estrogen-responsive human cancer cell lines (MCF7, T47D and Ishikawa). RESULTS: Our transcriptome analysis captured multiple estrogen-responsive genes and signaling pathways that increased cell proliferation, promoted DNA damage and genome instability, and decreased tumor suppressing effects, suggesting a common mechanism for estrogen-induced carcinogenesis. Comparative analysis revealed a core set of conserved estrogen-responsive genes that demonstrate enrichment of estrogen receptor binding sites and cell cycle signaling pathways. Knowledge-based and network analysis led us to propose that the mechanism involving estrogen-activated estrogen receptor mediated down-regulation of human homolog HES1 followed by up-regulation cell cycle-related genes (human homologs E2F4, CDK2, CCNA, CCNB, CCNE), is highly conserved, and this mechanism may involve novel crosstalk with basal AHR. We also identified mitotic roles of polo-like kinase as a conserved signaling pathway with multiple entry points for estrogen regulation. CONCLUSION: The findings demonstrate the use of zebrafish for characterizing estrogen-like environmental carcinogens and anti-estrogen drug screening. From an evolutionary perspective, our findings suggest that estrogen regulation of cell cycle is perhaps one of the earliest forms of steroidal-receptor controlled cellular processes. Our study provides first evidence of molecular conservation of estrogen-responsiveness between zebrafish and human cancer cell lines, hence demonstrating the potential of zebrafish for estrogen-related cancer research.

Genomewide expression profiling in the zebrafish embryo identifies target genes regulated by Hedgehog signaling during vertebrate development.

Hedgehog proteins play critical roles in organizing the embryonic development of animals, largely through modulation of target gene expression. Little is currently known, however, about the kinds and numbers of genes whose expression is controlled, directly or indirectly, by Hedgehog activity. Using techniques to globally repress or activate Hedgehog signaling in zebrafish embryos followed by microarray-based expression profiling, we have discovered a cohort of genes whose expression responds significantly to loss or gain of Hedgehog function. We have confirmed the Hedgehog responsiveness of a representative set of these genes with whole-mount in situ hybridization as well as real time PCR. In addition, we show that the consensus Gli-binding motif is enriched within the putative regulatory elements of a sizeable proportion of genes that showed positive regulation in our assay, indicating that their expression is directly induced by Hedgehog. Finally, we provide evidence that the Hedgehog-dependent spatially restricted transcription of one such gene, nkx2.9, is indeed mediated by Gli1 through a single Gli recognition site located within an evolutionarily conserved enhancer fragment. Taken together, this study represents the first comprehensive survey of target genes regulated by the Hedgehog pathway during vertebrate development. Our data also demonstrate for the first time the functionality of the Gli-binding motif in the control of Hedgehog signaling-induced gene expression in the zebrafish embryo.

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).