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)

Genomewide Expression Analysis in Zebrafish mind bomb Alleles with Pancreas Defects of Different Severity Identifies Putative Notch Responsive Genes

10.1371/journal.pone.0001479PLoS ONE3

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

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 (17β-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

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

10.1038/nbt1169Nature Biotechnology24173-75NABI

Hierarchical Clustering Analysis of Differentially Expressed Zebrafish Genes

<p>Of the 16,416 genes in the array, 3,657 genes, which showed a significant level of differential expression at least at one time point, were clustered hierarchically into groups on the basis of similarity of their expression profile, following Eisen's clustering method. The horizontal lines indicate the expression pattern of each gene, and the vertical rows indicate the embryonic developmental stages. For each gene, the ratio of transcript abundance in the developing stages of the embryo to its abundance in the reference RNA is represented by color intensities (red color indicates the higher expression, and green color indicates the lower expression of the gene in the embryos). Coordinated expression of the beta-actin gene (control) is indicated.</p

WISH Was Performed for Selected Clones

<p>All the clones subjected for in situ analysis showed a lower intensity of hybridization signal at one- to four-cell stages and increased intensity at 64-/128-cell and 256-/512-cell stages. These observations support the data obtained in the real-time PCR and microarray data, indicating the pre-MBT accumulation of transcripts.</p

Overview of the Expression Patterns of Genes Peaking at Selected Developmental Stages

<div><p>(A,B) Differentially expressed genes were clustered based on the peak of expression at the selected developmental stages and presented in the clustergram. Of the total number of 3,657 genes analyzed, 622, 609, 1,006, 688, and 732 genes showed peak expression at maternal, blastula, gastrula, segmentation, and pharyngula stages, respectively.</p><p>(C) General trend and average performance of the genes at each developmental stage are graphically represented (red, high expression; green, low expression). It is clear from this analysis that different sets of genes displayed their maximum at different stages of development, indicating the temporal/stage-specific maximum activity of the developmentally regulated genes.</p></div

Expression of Genes Involved in Specific Functions

<div><p>(A) Expression patterns of genes involved in the cell cycle. Genes involved in the cell cycle, namely cyclin and cyclin-dependent kinase genes, were identified based on GO and clustered separately. Most of the cyclin genes commenced their expression maternally, and the cyclin-dependent kinase genes showed expression throughout embryogenesis.</p><p>(B and C) Genes involved in ubiquitin function (proteasomes and ubiquitins) are active during most of the developmental stages, and the peak of activity is between the gastrula and segmentation stages.</p><p>(D) Gene expression during somitogenesis. Genes involved in somitogenesis were selected based on the GO list. Expression of MSP genes (acta 1, actc1, tpma, ckm, tnnt1, mibp2, myhz1, myhz2, mylz2, mylz3, tnnc, and tnnt3a) are clustered. Most of the MSP genes commenced their expression around 12 hpf and displayed their maximum during the pharyngula stage.</p><p>(E) Expression pattern of somitogenic (myotome-specific) transcription factors (mespa, mespb, mef2a, mef2c, med2d, myf5, myog, foxc1a, her4, her6, her7, and her9). Most of the transcription factors commenced transcript accumulation from the blastula stage onwards well in advance of commencement of MSP gene transcription.</p><p>(F) Coordinated expression of RP genes. From the array data, the expression profile of RP genes was clustered. Most of the genes showed an identical pattern of expression.</p></div