Figure 1. GR direct target genes show slight differences of expression between gr^ia30 and gr^s357 mutant lines. (A) RT-qPCR analysis of nr3c1 in 6 dpf gr^+/+1 and gr^ia30/ia30 larvae. (B) RT-qPCR analysis of nr3c1 in 6 dpf gr^+/+2 and gr^s357/s357 larvae. (C) RT-qPCR analysis of klf9 in 6 dpf gr^+/+1 and gr^ia30/ia30 (C) and in gr^+/+2 and gr^s357/s357 (C’) larvae with or without Dex treatment. (D) RT-qPCR analysis of epas1a in 6 dpf gr^+/+1 and gr^ia30/ia30 (D) and in gr^+/+2 and gr^s357/s357 (D’) larvae with or without Dex treatment. (E) RT-qPCR analysis of ucp2 in 6 dpf gr^+/+1 and gr^ia30/ia30 (E) and in gr^+/+2 and gr^s357/s357 (E’) larvae with or without Dex treatment. Statistical analyses were performed with Student’s t test. Mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2. Analysis of GC-dependent genes in mr mutant zebrafish larvae. (A) RT-qPCR analysis of nr3c2 in 6 dpf mr^+/+ and mr^ia32/ia32. (B) RT-qPCR analysis of nr3c1 in 6 dpf mr^+/+ and mr^ia32/ia32. (C) RT-qPCR analysis of nr3c2 in 6 dpf gr^+/+ and gr^ia30/ia30. (D) RT-qPCR analysis of nr3c2 in 6 dpf gr^+/+ and gr^s357/s357. (E) RT-qPCR analysis of klf9 in 6 dpf mr^+/+ and mr^ia32/iaq32 larvae with or without Dex treatment. (F) RT-qPCR analysis of epas1a in 6 dpf mr^+/+ and mr^ia32/ia32 larvae with or without Dex treatment. (G) RT-qPCR analysis of ucp2 in 6 dpf mr^+/+ and mr^ia32/ia32 larvae with or without Dex treatment. Statistical analyses were performed with Student’s t test. Mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3. GC-dependent genes reveal different expression levels in the two nr3c1 zebrafish mutant lines. (A) RT-qPCR analysis of ucp3 in 6 dpf gr^+/+1 and gr^ia30/ia30 (A) and in gr^+/+2 and gr^s357/s357 (A’) larvae with or without Dex treatment. (B) RT-qPCR analysis of ucp3 in 6 dpf mr^+/+ and mr^ia32/ia32 larvae with or without Dex treatment. (C) RT-qPCR analysis of slc25a25 in 6 dpf gr^+/+1 and gr^ia30/ia30 (C) and in gr^+/+2 and gr^s357/s357 (C’) larvae with or without Dex treatment. (D) RT-qPCR analysis of slc25a25 in 6 dpf mr^+/+ and mr^ia32/ia32 larvae with or without Dex treatment. Statistical analyses were performed with Student’s t test. Mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant.

Figure 4. Analysis of crosstalk between GR and Stat3 with zebrafish mutant and transgenic lines. (A) Representative pictures (A) and fluorescence quantification (A’) of Tg(7xStat3-Hsv.Ul23:EGFP)^ia28 6 dpf larvae at the level of the intestine in gr^+/+1, gr^+/ia30 and gr^ia30/ia30 genetic background. Scale bar= 100 μm. (B) Representative pictures (B) and fluorescence quantification (B’) of Tg(7xStat3-Hsv.Ul23:EGFP)^ia28 6 dpf larvae at the level of the intestine in gr^+/+2, gr^+/s357 and gr^s357/s357 genetic background. Scale bar = 100 μm. (C) Representative pictures (C) and fluorescence quantification (C’) of Tg(9xGCRE-Hsv.Ul23:EGFP)^ia20 incubated from 3 to 6 dpf with DMSO, 50 μM AG490 and 20 μM LIF. Scale bar = 500 μm. (D) representative pictures of 6 dpf larvae generated by the breeding between gr^+/ia30/stat3^+/ia23 zebrafish. Scale bar = 500 μm. (E) Table of observed (OV) and expected (EV) values of animals belonging to the 9 different genotypes obtained from breedings between gr^+/ia30/stat3^+/ia23 zebrafish: χ² test shows not significant differences between OV and EV in genotype distribution until 72 hpf (p-value = 0.7196); significant differences between OV and EV were detected at 4 dpf (** p-value = 0.0058) and 3 mpf (**** p-value = 4.30949 × 10⁻²²). Mean ± SEM. Statistical analyses were performed with Student’s t test (A,B,C) and χ² test (E). * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant.

Figure 5. Stat3-dependent genes are differentially expressed in gr^ia30 and gr^s357 mutant lines. (A) RT-qPCR analysis of socs3a in 6 dpf gr^+/+ and gr^ia30/ia30 (A) and in gr^+/+ and gr^s357/s357 (A’) larvae with or without Dex treatment. (B) RT-qPCR analysis of hif1αl in 6 dpf gr^+/+ and gr^ia30/ia30 (B) and in gr^+/+ and gr^s357/s357 (B’) larvae with or without Dex treatment. (C) RT-qPCR analysis of ulk2 in 6 dpf gr^+/+ and gr^ia30/ia30 (C) and in gr^+/+ and gr^s357/s357 (C’) larvae with or without Dex treatment. (D) RT-qPCR analysis of pnpla3 in 6 dpf gr^+/+ and gr^ia30/ia30 (D) and in gr^+/+ and gr^s357/s357 (D’) larvae with or without Dex treatment. (E) RT-qPCR analysis of ddit4 in 6 dpf gr^+/+ and gr^ia30/ia30 (E) and in gr^+/+ and gr^s357/s357 (E’) larvae with or without Dex treatment. (F) RT-qPCR analysis of socs3a in 6 dpf mr^+/+ and mr^ia32/ia32 larvae with or without Dex treatment. (G) RT-qPCR analysis of hif1αl in 6 dpf mr^+/+ and mr^ia32/ia32 larvae with or without Dex treatment. (H) RT-qPCR analysis of ddit4 in 6 dpf mr^+/+ and mr^ia32/ia32 larvae with or without Dex treatment. (I) RT-qPCR analysis of ulk2 in 6 dpf mr^+/+ and mr^ia32/ia32 larvae with or without Dex treatment. (J) RT-qPCR analysis of pnpla3 in 6 dpf mr^+/+ and mr^ia32/ia32 larvae with or without Dex treatment. Statistical analyses were performed with Student’s t test. Mean ± SEM. * p < 0.05; ** p< 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant.