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Global Expression Analysis of Vasculogenesis Induction by Zebrafish Ets1-Related Protein

ABSTRACT: Signaling pathways controlling vasculogenesis and angiogenesis are still poorly understood. Zebrafish Ets1-related protein (Etsrp) which encodes an ETS domain transcription factor, evolutionary related to the mammalian ER71 protein subfamily, has been identified as a major regulator of vasculogenesis and myelopoiesis and functions at the hemangioblast stage affecting the formation of both lineages. In the absence of Etsrp, angioblasts do not migrate or differentiate while overexpression of Etsrp results in the expansion of vascular endothelial and myeloid lineages. To identify genes functioning downstream of Etsrp we performed microarray analysis of etsrp-overexpressing embryos. Etsrp RNA injected embryos and control uninjected embryos were frozen at the tailbud stage and analyzed for expression of more than 30,000 genes using Nimblegen expression arrays. Approximately 300 genes showed greater than two-fold induction in etsrp-overexpressing embryos. Scl, crl, egfl7, aqp8, fli1a, fli1b, lmo2, cdh5 were among the previously known hemangioblast or vasculature-specific genes which were strongly upregulated in Etsrp-overexpressing embryos. We isolated and characterized a number of genes that were novel or previously unassociated with the zebrafish vasculature formation. Eight of them which include angiotensin II type 2 receptor (agtr2), src homology 2 domain containing E (she), similar to mannose receptor C1 (mrc1), endothelial cell-specific adhesion molecule (esam), cdc42 guanine nucleotide exchange factor 9 (arhgef9), yes-related kinase (yrk), zinc finger protein, multitype 2b (zfpm2b/fog2b) and stabilin 2 (stab2) were specifically expressed in vascular endothelial cells during early embryonic development while keratin18 expression was localized to the myeloid cells among others. Identification of novel vasculature and myeloid-specific genes that are regulated by Etsrp will be important for dissecting molecular mechanisms that regulate vasculogenesis / angiogenesis and myelopoiesis.

ORGANISM(S): Danio rerio

PROVIDER: GSE12909 | GEO | 2009/09/01

SECONDARY ACCESSION(S): PRJNA110817

REPOSITORIES: GEO

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Project description:Signaling pathways controlling vasculogenesis and angiogenesis are still poorly understood. Zebrafish Ets1-related protein (Etsrp) which encodes an ETS domain transcription factor, evolutionary related to the mammalian ER71 protein subfamily, has been identified as a major regulator of vasculogenesis and myelopoiesis and functions at the hemangioblast stage affecting the formation of both lineages. In the absence of Etsrp, angioblasts do not migrate or differentiate while overexpression of Etsrp results in the expansion of vascular endothelial and myeloid lineages. To identify genes functioning downstream of Etsrp we performed microarray analysis of etsrp-overexpressing embryos. Etsrp RNA injected embryos and control uninjected embryos were frozen at the tailbud stage and analyzed for expression of more than 30,000 genes using Nimblegen expression arrays. Approximately 300 genes showed greater than two-fold induction in etsrp-overexpressing embryos. Scl, crl, egfl7, aqp8, fli1a, fli1b, lmo2, cdh5 were among the previously known hemangioblast or vasculature-specific genes which were strongly upregulated in Etsrp-overexpressing embryos. We isolated and characterized a number of genes that were novel or previously unassociated with the zebrafish vasculature formation. Eight of them which include angiotensin II type 2 receptor (agtr2), src homology 2 domain containing E (she), similar to mannose receptor C1 (mrc1), endothelial cell-specific adhesion molecule (esam), cdc42 guanine nucleotide exchange factor 9 (arhgef9), yes-related kinase (yrk), zinc finger protein, multitype 2b (zfpm2b/fog2b) and stabilin 2 (stab2) were specifically expressed in vascular endothelial cells during early embryonic development while keratin18 expression was localized to the myeloid cells among others. Identification of novel vasculature and myeloid-specific genes that are regulated by Etsrp will be important for dissecting molecular mechanisms that regulate vasculogenesis / angiogenesis and myelopoiesis. Approximately 50 pg of etsrp RNA was microinjected into zebrafish embryos at the 1-4 cell stages. Three separate batches of approximately 150 microinjected embryos and uninjected controls were frozen on dry ice at the tailbud stage. Total RNA was purified using the RNAquous-4PCR kit (Ambion). Approximately 10-20 Âµg RNA from each batch was sent to the NimbleGen facility, were it was processed, labeled and hybridized to standard NimbleGen 385K gene expression array. Briefly, RNA was converted into cDNA using the SuperScript II cDNA Conversion Kit (Invitrogen, Carlsbad, CA). Double-stranded cDNA was random-prime labeled with Cy3-nonamers and hybridized to the microarrays for 16 hours at 42Â°C. The zebrafish microarray was based on Zv6 genome assembly and contained approximately 32,000 genes with approximately 12 probes of 60mer oligonucleotides per each gene. The arrays were washed, dried, and scanned at 5Î¼m resolution using a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA). Data were extracted from scanned images using NimbleScan software (Roche NimbleGen). Quantile normalization was performed across replicate arrays, and RMA (Robust Multichip Average) analysis was performed to generate gene expression values. Average ratios of expression values of etsrp-injected vs. control were calculated from three experiments. Results were sorted by the fold change from the highest to the lowest. Genes that displayed values of p>0.05 or showed expression levels less than 2-fold above the background level in both control and experimental samples were eliminated from further analysis.

Project description:Hemangioblasts are known as the common precursors for primitive hematopoietic and endothelial lineages. Their existence has been supported mainly by the observation that both cell types develop in close proximity and by in vitro differentiation and genetic studies. However, more compelling evidence will arise from tracking their cell fates using a lineage-specific marker. We report the identification of a hemangioblast-specific enhancer (Hb) located in the cis-regulatory region of chick Cerberus gene (cCer) that is able to direct the expression of enhanced green fluorescent protein (eGFP) to the precursors of yolk sac blood and endothelial cells in electroporated chick embryos. Moreover, we present the Hb-eGFP reporter as a powerful live imaging tool for visualizing hemangioblast cell fate and blood island morphogenesis. We hereby introduce the Hb enhancer as a valuable resource for genetically targeting the hemangioblast population as well as for studying the dynamics of vascular and blood cell development. We used microarray analysis of Hb-eGFP expressing cells to verify the expression of hemangioblast-specific genes in this cell population. In order to characterize the gene expression profile of the Hb-eGFP-positive population, we have isolated Hb-eGFP-positive cells and compared their expression profile with the control RFP-positive population. In brief, chicken embryos were electroporated at HH3 with Hb-eGFP and pCAGGS-RFP reporter constructs, observed under a fluorescence stereoscope and harvested at stage HH5-6 into three groups of four embryos each. Embryos were dissociated into single cell suspensions and the eGFP-positive and eGFP-negative/RFP-positive cell populations were sorted by Fluorescence Activated Cell Sorting (FACS). Cell populations were collected simultaneously into two different tubes, containing RNAlater (Ambion) and subsequently used for RNA extraction. Total RNA was isolated from triplicates of each population, evaluated for RNA integrity, reverse transcribed, amplified and hybridized against six Affymetrix Chicken Genome microarrays.

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Global Expression Analysis of Vasculogenesis Induction by Zebrafish Ets1-Related Protein

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