Abstract : The 11b-hydroxylase (11bH) is the enzyme responsible for the biosynthesis of cortisol, the main corticosteroid in fish, which is implicated in the regulation of multiple physiological processes including energy metabolism, hydromineral balance, reproduction and the immune system. In fish, the gene coding for 11bH, namely cyp11c1, is expressed early in interrenal cells during the development participating to the emergence of the corticosteroid axis. To further investigate the expression cyp11c1 and its potential deregulation by stressors and pharmaceutical ligands, we established a transgenic zebrafish line that expresses Green Fluorescent Protein (GFP) under the control of the zebrafish cyp11c1 promoter. Using in vivo fluorescence imaging techniques on whole larvae, we showed a weak expression of the reporter gene from 4 to 7 days post-fertilization (dpf) in an anatomical region where interrenal cells are known to be present. Tissue sections and immunostaining with specific 11bH antibodies revealed that GFP is co-localized with the endogenous cyp11c1 protein in interrenal cells. This well agrees with the expression and localization of other steroidogenic interrenal marker genes such as star and cyp11a1 in wild-type zebrafish at this stage of development (To et al. 2007). To further characterize the cyp11c1-GFP zebrafish model, GFP was quantified in 6-dpf old zebrafish after osmotic stress or exposure to glucocorticoids receptor (GR) agonists (Dexamethasone, DEX) and antagonists (Mifepristone, MIF). Exposure to salt water led to a rapid induction of GFP, showing that cyp11c1 responds to an osmotic shock. Exposure to MIF (1µM from 5 to 6 dpf) decreases GFP intensity, suggesting that GR is involved in the transcriptional regulation of cyp11c1 during development. In DEX-exposed larvae (10µM, 5-6dpf), GFP intensity was inhibited as compared to controls, this effect being abolished when zebrafish larvae were co-exposed with MIF. These data agree with the known negative feedback of DEX on pituitary expression of Pro-opiomelanocortin (POMC), leading in turn to a down-regulation of genes involved in corticosteroidogenesis (Sun et al., 2010). Altogether, these data suggest that the newly developed transgenic cyp11c1-GFP zebrafish is an interesting model to study the expression of 11b-hydroxylase gene in interrenal cells and its perturbation by endocrine active compounds acting on the Hypothalamic-Pituitary-Interrenal axis, which is critical for cortisol homeostasis.
