Defl1 knockdown gene expression in Zebrafish 12.5 hpf embryos
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ABSTRACT:
ORGANISM(S): Danio rerio
SUBMITTER: Graeme Grimes
PROVIDER: E-GEOD-31766 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2008-08-12 | E-GEOD-12039 | biostudies-arrayexpress
Project description:Fish, JE, Santoro, MM, Morton, SU, Yu, S, Yeh, RF, Wythe, JD, Ivey, KI, Bruneau, BG, Stainier, DYR, and Srivastava, D. (2008). miR-126 Regulates Angiogenic Signaling and Vascular Integrity. Developmental Cell 15, 272-284. Precise regulation of the formation, maintenance, and remodeling of the vasculature is required for normal development, tissue response to injury, and tumor progression. How specific microRNAs intersect with and modulate angiogenic signaling cascades is unknown. Here, we identified microRNAs that were enriched in endothelial cells derived from mouse embryonic stem (ES) cells and in developing mouse embryos. We found that miR-126 regulated the response of endothelial cells to VEGF. Additionally, knockdown of miR-126 in zebrafish resulted in loss of vascular integrity and hemorrhage during embryonic development. miR-126 functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1 and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-β). Increased expression of Spred1 or inhibition of VEGF signaling in zebrafish resulted in defects similar to miR-126 knockdown. These findings illustrate that a single miRNA can regulate vascular integrity and angiogenesis, providing a new target for modulating vascular formation and function. Experiment Overall Design: To identify the genes regulated by miR-126 in vivo, flk1:GFP transgenic zebrafish embryos (which express GFP in the endothelium) were injected at the one-cell stage with 4 ng of two independent antisense morpholinos that block the processing of the miR-126 pri-cursor. At 48 hours post-fertilization, endothelial cells were isolated by fluorescence-activated cell sorting from flk1:GFP transgenic fish, and RNA was extracted. Arrays were performed on four biological replicates of control and two independent miR-126 morpholinos.
2008-10-25 | E-GEOD-12012 | biostudies-arrayexpress
Project description:The Spi1/ Pu.1 transcription factor plays a crucial role in myeloid cell development across many species. Several Spi1 target genes have been identified so far, yet the Spi1-dependent gene group remains largely unknown. To identify novel genes downstream of Spi1 we employed a microarray strategy using zebrafish embryos. We established the gene group down-regulated upon spi1 knockdown while simultaneously enriched in FACS-sorted embryonic myeloid cells of a spi1:GFP transgenic line, thus representing putative myeloid-specific Spi1 target genes. This gene group contained all previously identified Spi1-dependent zebrafish genes, confirming the validity of the approach, as well as novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes cxcr3.2, mpeg1, ptpn6 and mfap4 were expressed specifically in early embryonic macrophages. The analysis of adult zebrafish hematopoietic tissue showed that genes mfap4 and mpeg1 remained macrophage specific within the myeloid fraction throughout zebrafish life. We also demonstrated that gene cxcr3.2, coding for chemokine receptor 3.2, functions in macrophage migration to the site of bacterial infection. These results establish a myeloid-specific gene group dependent on Spi1 in zebrafish and identify novel early macrophage-specific marker genes, which will facilitate further studies of macrophage development and innate immune function. Zebrafish strains Zebrafish (Danio rerio) were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). The spi1-gfp transgenic line used in this study (Pu1-lynEGFP, a gift from Franscesca Peri, EMBL, Heidelberg) contains 4 kb of spi1/pu.1 promoter sequence driving expression of membrane-targeted EGFP. Morpholino knock-down experiments Morpholino oligonucleotides (Gene Tools) were diluted to required concentration in 1x Danieu’s buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES; pH 7.6) containing 1% Phenol red (Sigma) and approximately 1 nl was injected into the 1-2 cell stage embryo using a Femtojet injector (Eppendorf). Embryo dissociation and FACS (Fluorescent activated cell sorting) Dissociation of embryos was performed according to Covassin et al. 30. In short, ~300 embryos of spi1:GFP line at 28 hpf were dechorionated by pronase treatment, rinsed in calcium free Ringer solution, followed by digestion with 0.25% trypsin. The obtained cell suspension was centrifuged, rinsed with PBS and resuspended in Leibovitz medium L15 without phenol red, 1% fetal calf serum, 0.8mM CaCl2, penicillin 50 U/ml and streptomycin 0.05 mg/ml. The single cell suspension was subject to FACS at room temperature using a FACSAria (Becton Dickinson) with the BD FACSDiva software version 5.0.3 and a Coherent Sapphire solid state laser 488 nm with 13 mW power. The GFP+ and GFP- cell fractions were collected separately into L15 medium supplemented with 0.8 mM CaCl2, 10% fetal calf serum and penicillin 50 U/ml and streptomycin 0.05 mg/ml. The standard yield from circa 300 embryos was approximately 2 x 105 GFP+ cells. Microarray experiment design and analysis Embryos injected either with 1 mM Pu1 morpholino19 or 1 mM Standard Control morpholino were harvested at 28 hours post fertilization (hpf). As a control, embryos injected with an equal volume of 1% Phenol red in Danieu’s buffer (1xPR/D) were used. Pools of 20-30 embryos were collected. RNA samples from spi1 morphants and standard control morphants were labelled with Cy5 and hybridized against a Cy3-labelled common reference (a mixture of all samples injected with 1xPR/D). This experiment was performed in triplicate. FACS sorted spi1-GFP zebrafish embryos at 28hpf were analyzed as follows: the RNA of GFP-positive fraction was labelled with Cy5 and hybridized against a Cy3-labelled RNA of corresponding GFP-negative fraction of cells from the same pool of embryos. This experiment was performed in duplicate. RNA isolation, synthesis of amino allyl labeled aRNA, coupling of Cy3 and Cy5 dyes, and hybridization conditions were as described31. Microarray analysis was performed using custom-designed 44k Agilent chips (platform accession number in GEO: GPL7735) described elsewhere 31. Microarray data were processed and analyzed as described 31. Significance cut-offs for differentially expressed probe sequences were set at 1.2 fold change at P <10-2.
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