Glucocorticoid receptor, but not mineralocorticoid receptor, mediates cortisol regulation of epidermal ionocyte development and ion transport in zebrafish (danio rerio).

Cortisol is the major endogenous glucocorticoid (GC) both in human and fish, mediated by corticosteroid receptors. Due to the absence of aldosterone production in teleost fish, cortisol is also traditionally accepted to function as mineralocorticoid (MC); but whether it acts through the glucocorticoid receptor (GR) or the mineralocorticoid receptor (MR) remains a subject of debate. Here, we used loss-of-function and rescue assays to determine whether cortisol affects zebrafish epidermal ionocyte development and function via the GR and/or the MR. GR knockdown morphants displayed a significant decrease in the major ionocytes, namely Na(+)-K(+)-ATPase-rich cells (NaRCs) and H(+)-ATPase-rich cells (HRCs), as well as other cells, including epidermal stem cells (ESCs), keratinocytes, and mucus cells; conversely, cell numbers were unaffected in MR knockdown morphants. In agreement, GR morphants, but not MR morphants, exhibited decreased NaRC-mediated Ca(2+) uptake and HRC-mediated H(+) secretion. Rescue via GR capped mRNA injection or exogenous cortisol incubation normalized the number of epidermal ionocytes in GR morphants. We also provide evidence for GR localization in epidermal cells. At the transcript level, GR mRNA is ubiquitously expressed in gill sections and present in both NaRCs and HRCs, supporting the knockdown and functional assay results in embryo. Altogether, we have provided solid molecular evidence that GR is indeed present on ionocytes, where it mediates the effects of cortisol on ionocyte development and function. Hence, cortisol-GR axis performs the roles of both GC and MC in zebrafish skin and gills.

Cortisol promotes differentiation of epidermal ionocytes through Foxi3 transcription factors in zebrafish (Danio rerio).

Glucocorticoid regulates epidermal cell proliferation, and is used to treat certain skin disorders. Cortisol, a glucocorticoid, is also linked to skin development in teleost fish. Cortisol increases the number of epithelial ionocytes during environmental acclimation in euryhaline fishes, but it is unclear whether this is due to increased differentiation or proliferation. To investigate, we treated zebrafish embryos with exogenous cortisol (20mg/L). The densities of the ionocytes Na(+)-K(+)-ATPase rich cells (NaRCs) and H(+)-ATPase rich cells (HRCs) were significantly increased by cortisol, and this was accompanied by an increase in the respective marker genes. Expression of the glucocorticoid receptor (GR) gene was decreased. Cortisol treatment also increased ionocytes in cultured adult zebrafish gills, and up-regulated expression of genes encoding forkhead box I3 (foxi3a and foxi3b) transcription factors, which regulate ionocyte progenitor development. GR expression was up-regulated by cortisol in vitro; as such, the observed decrease in vivo reflects a regulatory systemic-negative feedback. Notably, in situ hybridization revealed that foxi3a/b mRNA expression was increased by cortisol at 24-48h post-fertilization. Cortisol also decreased keratinocytes, but did not affect epidermal stem cells or mucus cells. We conclude that foxi3a/b transactivation by cortisol-GR favors differentiation of ionocyte progenitors, thereby facilitating proliferation of mature ionocytes.