A new CRISPR-Cas9-induced mutation of the zebrafish GR. (A) Schematic of the nr3c1 gene and encoded GR showing location of the targeted sequence and resulting premature stop codon with respect to the protein domains: N-terminal domain (NTD), DNA binding domain (DBD), and ligand binding domain (LBD). (B) Nucleotide sequence of nr3c1 exon 3, showing sequence targeted by the gRNA (underlined), the 20 base deletion resulting from injection of that gRNA and Cas9 mRNA (gray), and the resulting premature stop codon (bold italic). (C) Predicted amino acid sequence of the full-length zebrafish GR, plus the extra amino acids introduced by the frameshift shown in (B) (red font). The sequence eliminated by the premature stop is shown in gray, with the DBD underlined. Methionines corresponding to potential alternative initiation sites are shown in green. The positions of all reported frameshift mutations (including that reported here, E369) are boxed. The arginine that is changed to a cysteine in the gr^s357 mutation is shown in orange. (D) Schematic of the GR showing locations of the two previously reported exon 2 frameshift mutations resulting in truncations at amino acids 178 and 310, and the one in exon 3 reported here truncating at amino acid 369, and residues included in N310 and N369 mRNA constructs produced for microinjection. (E) Relative expression of fkbp5 in 6 hpf homozyogous GR^369- embryos, either uninjected or injected with mRNA encoding full-length GR (GR-FL), N-terminally truncated GR isoforms (GR-N310 and GR-N369), or a nonspecific control mRNA encoding Xenopus elongation factor 1α (Xeno EF).

RNA-seq identifies GR-dependent and GR-independent genes and effects of chronic cortisol treatment. (A) Diagram of experimental design using Visual Background Adaptation (VBA) selection. (B) Principal component plot of the RNA-seq data, showing the location of each replicate sample with respect to the first two principal components. Cortisol-treated samples are shown in red, vehicle-treated controls in black. VBA+ samples (containing at least one functional GR allele) are filled shapes, VBA- samples are empty shapes. (C) Venn diagram showing numbers of genes upregulated in each of the indicated comparisons (keyed as in (A)): VBA+ vs. VBA- (vehicle-treated controls); cortisol-treated vs. vehicle-treated VBA+; and cortisol-treated vs. vehicle treated VBA-. Upregulated genes in common respectively between the first two and the last two comparisons are listed on the right. (D) Box plots of Z-transformed expression levels of klf9 and npas4a obtained from the RNA-seq data. Sample numbers correspond to those in (B). Asterisks indicate significant differential expression (adjusted p values): ***< .0001; *< .05.

CRISPR-Cas9-mediated disruption of zebrafish klf9. (A) Schematic of the klf9 locus and encoded protein showing location of the gRNA target in exon 1, which introduces a premature stop codon upstream of the DNA binding domain. (B) Sequence of klf9 exon 1, with 5′ untranslated region in small case and coding sequence in upper case. The location of the 2 base pair deletion generated by CRISPR is shown in gray font; the gRNA target sequence is underlined; and the premature stop codon introduced by frameshift is noted with an asterisk. (C) Effect of chronic cortisol treatment and the frameshift mutation on relative levels of klf9 mRNA and pre-mRNA, measured by qRT-PCR. The results show the averages and SEM of three biological replicates; significance values were obtained by ANOVA.

RNA-seq identifies klf9-dependent transcriptomic effects of chronic cortisol exposure. (A) Scatter plot comparing differential gene expression in response to chronic cortisol treatment in wild type (WT) and klf9^-/- larvae. (B) Venn diagram comparing numbers of genes upregulated by cortisol treatment in wild type and klf9^-/- larvae, and overlap. (C) Examples of two of the genes upregulated by chronic cortisol in a klf9-dependent fashion that were also identified in our previous analysis. (D) Treemap generated by REVIGO⁵⁷ (https://revigo.irb.hr/) of gene ontology biological process terms found by GOrilla³⁴ to be enriched in the set of 408 genes upregulated by chronic cortisol in a klf9-dependent way.

Combined analysis of three different RNA-seq datasets examining the transcriptomic effects of chronic cortisol exposure. (A) Principal component plot showing the location of each wild-type or VBA+ sample from all three RNA-seq experiments with respect to PCs 4 and 5. (B) Treemap generated by REVIGO⁵⁷ (https://revigo.irb.hr/) of GO biological process terms found by GOrilla³⁴ to be associated with PC5 ranked by upregulation in response to chronic cortisol treatment.

Identification and analysis of a common set of genes upregulated by chronic cortisol treatment in multiple RNA-seq experiments. (A) Venn diagrams showing numbers of upregulated and downregulated genes following reanalysis of the data from all three experiments and their overlap. Only 8 of the 12 genes in common between all experiments were annotated with gene names (listed). (B) Violin and box plots of estimated fold change of the 149 genes that are upregulated in at least 2 of the three experiments shown in (A); the differences between each experiment are statistically significant (Table S14). (C) Treemap generated by REVIGO⁵⁷ (https://revigo.irb.hr/) of GO biological process terms found by GOrilla³⁴ to be associated with the 149 genes upregulated by chronic cortisol treatment in at least two out of the three experiments.

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