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, GR^369-, 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 GR^369- 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.

斑马鱼最近成为研究糖皮质激素信号的发育作用和糖皮质激素诱导的发育程序的潜在机制的模型系统。为了评估糖皮质激素受体 (GR) 在此类编程中的作用，我们使用 CRISPR-Cas9 产生新的移码突变 GR ^369-，这消除了 DNA 结合域上游的所有潜在的框内起始密码子。使用 RNA-seq 询问这种突变如何在正常条件下和慢性皮质醇治疗下影响幼虫转录组，我们发现 GR 介导了治疗的大部分效果，而且矛盾的是，皮质醇治疗的幼虫的转录组更像缺乏 GR 的幼虫与具有 GR 的幼虫相比，表明经皮质醇处理的幼虫产生了 GR 抗性。一种在 GR ^369-幼虫中表达不足并在皮质醇处理的幼虫中持续过度表达的转录调节因子是klf9。因此，我们使用了 CRISPR-Cas9 介导的klf9突变和 RNA-seq 以评估正常和皮质醇处理的幼虫中 Klf9 依赖性基因表达。我们的结果表明 Klf9 对慢性皮质醇暴露的转录组反应有显着贡献，介导了我们之前报道的促炎基因的上调。