Abstract
The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR369-, which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR369- larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously.
Highlights
The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming
Homozygous G R369- males crossed with wild-type females produced viable embryos, whereas most homozygous GR369- females crossed with either mutant or wild-type males produced embryos that all died within 24 h of fertilization
The set of genes upregulated by cortisol in wild-type but not klf9-/- embryos included four interferon regulatory factors, two interleukins, and four interleukin receptors. These results indicate that Klf[9] contributes in a significant way to the proinflammatory gene expression induced by chronic cortisol exposure
Summary
The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. We report a new loss-of-function mutation, consisting of a deletion in nr3c1 exon 3 that results in a frameshift and premature stop codon immediately upstream of the DNA binding domain which eliminates GR activity as a transcriptional activator Using this mutant line, we performed an RNA-seq experiment to identify genes regulated by the GR and to parse GR-dependent and GR-independent effects of chronic cortisol treatment on larval gene expression. We found that most of the transcriptomic effects of the chronic cortisol treatment are mediated by the GR and identified klf[9], which encodes a ubiquitously expressed[16] member of the krüppel-like family of zinc finger transcription factors, as the one regulatory gene consistently upregulated by the GR under both normal conditions and in response to chronic cortisol treatment. Our results indicate that klf[9] mediates the cortisol-induced upregulation of pro-inflammatory genes that we reported previously[9], and suggest that klf[9] is a core feedforward regulator of the transcriptional response to glucocorticoid signaling
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