6 research outputs found

    Confirmation of microarray findings by qPCR analysis.

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    <p>qPCR analysis was performed on 7 genes to confirm the transcript abundance seen with the microarray analysis. The selected genes were <i>bmp7a</i> (A), <i>f5</i> (B)<i>, ff1d</i> (C), <i>myom1a</i> (D), <i>pomca</i> (E), star (F), <i>mc1r</i> (G). Data is presented as mean ± standard error of the mean (normalized to β-actin, SEM; n=5-7 pools of 25 embryos each); * denotes statistical significance (<i>t</i>-test, p<0.05). (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080726#pone-0080726-t007" target="_blank">Table 7</a> for fold-changes and p-values).</p

    Distribution of fold-change for statistically significant genes at 24 and 36 hpf in response to GR knockdown.

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    <p>This figure presents the frequency of genes that were up- or down-regulated at specific fold-change ranges at 24 hpf (A) or 36 hpf (B). In general, there was a relatively normal distribution at 24 hpf, with relatively few genes showing extreme fold changes and a balance between up and downregulation. At 36 hpf, a far greater percentage of genes were upregulated than downregulated, and none showed reduction as severe as at 24 hpf.</p

    Interactome networks of select pathways identified as GR responsive by Ingenuity Pathway Analysis software.

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    <p>The IPA software organized and classified genes to identify important networks that were modulated by GR knockdown. These interactome networks detail the regulatory connections between genes as detailed by the connective arrows. The most strongly affected process was nervous system development at both 24 hpf (A) and 36 hpf (B). Other interesting pathways are DNA replication and energy production at 24 hpf (C), cardiovascular development at 36 hpf (D) and developmental disorders at 36 hpf (E). These final 3 pathways each involve a gene that were quantified by qPCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080726#pone-0080726-g004" target="_blank">Figure 4</a>): <i>f5</i> (C, E), and <i>pomca</i> (D). Single-way arrows indicate one gene regulating another, two-sided arrows indicate co-regulation, looped arrows indicate self-regulation. The shape of each member of the network indicates its cellular location (according to IPA software classifications): Extracellular (diamond); plasma membrane (hexagon); cytosol (square); nucleus (circle); unknown (triangle). The color of each member of the network indicates its mean fold change range: >2 (dark green); 1-2 (light green); unchanged (grey); 0.5-1 (light red), <0.5 (dark red).</p

    Functional annotation (using Ingenuity Pathway Analysis software) of genes that were upregulated and downregulated by GR knockdown.

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    <p>Ingenuity pathway analysis software identified prominent developmental pathways that were significantly affected by GR knockdown based on the significantly changed genes at 24 hpf (A) and 36 hpf (B). Each pathway is named and the total number of genes as well as the number of upregulated and downregulated genes is listed (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080726#pone-0080726-t005" target="_blank">Tables 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080726#pone-0080726-t006" target="_blank">6</a> for complete list of genes).</p

    Numbers of statistically significant genes upregulated and downregulated at 24 and 36 hpf in response to GR knockdown.

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    <p>Of 12261 potential unique genes, the mRNA expression of 1313 were found to be statistically significantly changed at 24 hpf, with 583 downregulated (grey) and 730 upregulated (black). 836 genes were changed with statistical significance at 36 hpf, of which 243 were downregulated and 593 were upregulated. (n=3 pools of 25 embryos used for microarray analysis, P≤0.05, Students <i>t</i>-test with Benjamini-Hochberg false-discovery rate correction).</p

    The Transcriptomics of Glucocorticoid Receptor Signaling in Developing Zebrafish

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    <div><p>Cortisol is the primary corticosteroid in teleosts that is released in response to stressor activation of the hypothalamus-pituitary-interrenal axis. The target tissue action of this hormone is primarily mediated by the intracellular glucocorticoid receptor (GR), a ligand-bound transcription factor. In developing zebrafish (<i>Danio rerio</i>) embryos, GR transcripts and cortisol are maternally deposited into the oocyte prior to fertilization and influence early embryogenesis. To better understand of the molecular mechanisms involved, we investigated changes in the developmental transcriptome prior to hatch, in response to morpholino oligonucleotide knockdown of GR using the Agilent zebrafish microarray platform. A total of 1313 and 836 mRNA transcripts were significantly changed at 24 and 36 hours post fertilization (hpf), respectively. Functional analysis revealed numerous developmental processes under GR regulation, including neurogenesis, eye development, skeletal and cardiac muscle formation. Together, this study underscores a critical role for glucocorticoid signaling in programming molecular events essential for zebrafish development.</p> </div
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