Zebrafish transcriptomics in 24 and 36 hpf embryos after knockdown of glucocorticoid receptor translation
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ABSTRACT:
ORGANISM(S): Danio rerio
SUBMITTER: Matt Vijayan
PROVIDER: E-GEOD-50376 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The glucocorticoid receptor (GR) regulates gene expression upon activation by glucocorticoid (GC) hormones. In zebrafish, two GR splice variants exist: the canonical GR M-NM-1-isoform (GRM-NM-1), and the GR M-NM-2-isoform (GRM-NM-2). The exact function of GRb remains elusive. We have investigated the transcriptional role of GRa and GRb in the zebrafish embryo model by injecting mebryos with two splice-blocking morpholinos (one leading to knockdown of both GR M-NM-1- and M-NM-2-isoforms, and another targeting the alternative splicing of the GR pre-mRNA in favor of the GR M-NM-2-isoform) and with GRM-NM-2 mRNA (resulting in specific GRM-NM-2 overexpression). Transcriptome profiling was performed on total RNA isolated from 30 hpf embryos. Our results show that GRb does not act as a dominant-negative inhibitor of GRa, and that GRa regulates two distinct gene clusters, which are mainly involved in the metabolism of the embryo. This microarray study was designed to determine the effect of knockdown and overexpression of GRa and GRb. For this purpose, wild type (AB/TL) zebrafish embryos were injected at the 1-2 cell stage with a standard control morpholino (SC-MO), a splice-blocking morpholino leading to knockdown of both GR M-NM-1- and M-NM-2-isoforms (MO1), a splice-blocking morpholino targeting the alternative splicing of the GR pre-mRNA in favor of the GR M-NM-2-isoform (MO2), or GRb mRNA. At 24 hpf, each group was treated with dexamathasone (or vehicle) for 6 hr. This resulted in 8 treament groups: SC-MO/veh, SC-MO/dex, MO1/veh, MO1/dex, MO2/veh, MO2/dex, GRb mRNA/veh, GRb mRNA/dex. After the incubation period, embryos were collected and RNA was isolated from 20 embryos per treatment group (30 hpf). Three individual experiments were performed, and the resulting triplicate samples were analyzed by microarray, using a common reference approach.
2014-11-18 | E-GEOD-63360 | biostudies-arrayexpress
Project description:In zebrafish, ovulated oocytes contain both cortisol deposited from the maternal circulation and maternal mRNA for the glucocorticoid receptor (gr mRNA), which is spread as granular structures throughout the central ooplasm. At the 1-cell stage (0.2 hpf), this transcript is relocated by streamers in the blastodisc area and equally partitioned among blastomeres. At 15 hpf, it is replaced by the zygotic transcript. Morpholino knockdown was applied to block translation (grATG1MO or MO2-nr3c1 and grATG2MO or MO3-nr3c1) of both maternal and zygotic gr transcripts, while a missplicing morpholino (grmismMO or MO4-nr3c1) was used to block post-transcriptionally the zygotic transcript alone. MO2-nr3c1 and MO3-nr3c1 (but not MO4-nr3c1) treatment produced craniofacial and caudal malformations in 1-dpf embryos and 5-dpf larvae, which were also affected by pericardial oedema, persistent yolk sac, reduced subintestinal veins, altered neurogenesis and uninflated swim bladder. Such effects were rescued with trout gr2 mRNA. Pangenomic microarray analysis revealed that 114 and 37 highly expressed transcripts were up- and down-regulated, respectively, by maternal GR protein deficiency in 5-hpf embryos. Similar alterations were found at 10 hpf. These effects were confirmed by real-time PCR of 2 up- (casp8, grp1 and igf2a) and 1 down-regulated transcripts (mcm6) evaluated at 4, 8 and 12 hpf. As the contents of transcripts were modified already at 4 hpf, it seems that the lack of GR affects both ways the molecular machinery for the degradation of maternal mRNAs. These results indicate that the maternal gr transcript participates in the maternal programming of zebrafish development. MO2-nr3c1 morphants were compared with MO2-nr3c1-5m morphants at 5 hpf and 10 hpf. MO2-nr3c1 morphants were compared with wild type (WT) at 5 hpf and 10 hpf. MO2-nr3c1 is a morpholino selected to knockdown translation of gr mRNA. MO2-nr3c1-5m is a specific control morpholino.
2011-11-16 | E-GEOD-25517 | biostudies-arrayexpress
Project description:Transcriptional profiling of zebrafish embryo comparing wild type untreated embryos with embryos injected with morpholino of zf-bad. This assay is used for determination of expression profiling at 24 hpf and 48 hpf under Bad deficiency. Two-condition experiment, wild type vs. MO-Bad treated cells. Biological replicates: each group contains 200 embryos.
2014-06-01 | E-GEOD-47971 | biostudies-arrayexpress
Project description:Macrophage expressed gene 1 (MPEG1) encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates, MPEG1 is an integral membrane protein of macrophages, but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1, two of which (mpeg1 and mpeg1.2) are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens, Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down-regulated during infection with both pathogens, mpeg1.2 is infection inducible. Up-regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1, and requires the Toll-like receptor adaptor molecule MyD88 and transcription factor NFM-NM-:B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection, both mpeg1 and mpeg1.2 knockdown increase bacterial burdens, but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti-bacterial function of the Mpeg1 family and indicate that the intricate cross-regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Embryos were injected at the one cell stage with a morpholino targeting mpeg1, or with the standard control morpholino from GeneTools, or with a morpholino targeting ptpn6 (Kanwal et al., 2013, J. Immunol 190:1631-45) for comparison. Subsequently, at 24 hours post fertilisation (hpf) the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein, or mock-injected with PBS/2%PVP. After injections embryos were transferred into fresh egg water containing 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich) to prevent melanisation and incubated for 4 days at 28M-BM-0C. After the incubation period, infected and uninfected morphants, mutants and their controls were imaged and groups of 30 embryos were snap-frozen in liquid nitrogen and RNA was isolated for Illumina RNAseq analysis.
2014-09-01 | E-GEOD-54885 | biostudies-arrayexpress
Project description:Deficiency in the protein-tyrosine phosphatase SHP1/PTPN6 is linked with hematological malignancies and chronic inflammatory diseases. Here we exploited the embryonic and larval stages of zebrafish as an animal model to study ptpn6 function in the sole context of innate immunity. We show that ptpn6 knockdown induces a spontaneous inflammation-associated phenotype at the late larval stage, which was microbe-independent and enhanced instead of suppressed by glucocorticoids. When challenged with Salmonella typhimurium or Mycobacterium marinum at earlier stages of development, the innate immune system was hyperactivated to a contra-productive level that impaired the control of these pathogenic bacteria. These results demonstrate the crucial regulatory function of ptpn6 in preventing host-detrimental effects of inflammation by imposing a tight control over the level of innate immune response activation. This microarray study was designed to determine the effect of morpholino knockdown of the ptpn6 gene on the innate immune response of zebrafish embryos during infection with Salmonella typhimurium. Three independent infection experiments were performed using mixed egg clutches of wild type zebrafish, which were a cross of the AB and TL strains. Each experiment consisted of the following four treatment groups: (1) uninfected control embryos, (2) S. typhimurium-infected control embryos, (3) uninfected ptpn6 knockdown embryos, and (4) S. typhimurium-infected ptpn6 knockdown embryos. Knockdown of ptpn6 was performed by micro-injecting embryos at the 1-2 cell stage with the ptpn6 morpholino, and control embryos were mock-injected with buffer. Embryos were grown at 28.5M-bM-^@M-^S30M-BM-0C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200-250 colony forming units (CFU) of S. typhimurium SL1027 bacteria into the caudal vein, or were mock-injected with buffer as a control. After injections embryos were transferred into fresh egg water and incubated for 8 h at 28M-BM-0C. After the incubation period, pools of 15-20 embryos per treatment group were snap-frozen in liquid nitrogen and RNA was isolated for microarray analysis. All treatment groups were analyzed using a common reference approach.
2012-12-31 | E-GEOD-34930 | biostudies-arrayexpress
Project description:Transcriptional profiling of zebrafish embryos comparing wild type untreated embryos with embryos injected with morpholino of zf-grna. This assay is used for the determination of expression profiling at 22 hpf under GRN-A deficiency. Two-condition experiment: wild type vs. MO-grnA treated cells. 3 biological replicates: each group contains 200 embryos.
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Project description:To further characterize the roles of cortisol signaling via the glucocorticoid receptor (GR) in developing zebrafish, we have used morpholino oligonucleotides to knockdown GR protein translation and measured gene expression in RNA extracted from 24 and 36 hours post fertilization (hpf) embryos. The GR morpholino was characterized previously in Nesan et al., 2012, Endocrinology 181, 35-44)
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2010-08-01 | E-GEOD-21024 | biostudies-arrayexpress
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2013-10-09 | E-GEOD-45699 | biostudies-arrayexpress