Aquaporin 1 Is Involved in Acid Secretion by Ionocytes of Zebrafish Embryos through Facilitating CO2 Transport.

Mammalian aquaporin 1 (AQP1) is well known to function as a membrane channel for H2O and CO2 transport. Zebrafish AQP1a.1 (the homologue of mammalian AQP1) was recently identified in ionocytes of embryos; however its role in ionocytes is still unclear. In this study, we hypothesized that zebrafish AQP1a.1 is involved in the acid secretion by ionocytes through facilitating H2O and CO2 diffusion. A real-time PCR showed that mRNA levels of AQP1a.1 in embryos were induced by exposure to 1% CO2 hypercapnia for 3 days. In situ hybridization and immunohistochemistry showed that the AQP1a.1 transcript was highly expressed by acid-secreting ionocytes, i.e., H+-ATPase-rich (HR) cells. A scanning ion-selective electrode technique (SIET) was applied to analyze CO2-induced H+ secretion by individual ionocytes in embryos. H+ secretion by HR cells remarkably increased after a transient loading of CO2 (1% for 10 min). AQP1a.1 knockdown with morpholino oligonucleotides decreased the H+ secretion of HR cells by about half and limited the CO2 stimulated increase. In addition, exposure to an AQP inhibitor (PCMB) for 10 min also suppressed CO2-induced H+ secretion. Results from this study support our hypothesis and provide in vivo evidence of the physiological role of AQP1 in CO2 transport.

Stanniocalcin-1 controls ion regulation functions of ion-transporting epithelium other than calcium balance.

Stanniocalcin-1 (STC-1) was first identified to involve in Ca(2+) homeostasis in teleosts, and was thought to act as a hypocalcemic hormone in vertebrate. Recent studies suggested that STC-1 exhibits broad effects on ion balance, not confines to Ca(2+), but the mechanism of this regulation process remains largely unknown. Here, we used zebrafish embryos as an alternative in vivo model to investigate how STC-1 regulates transepithelial ion transport function in ion-transporting epithelium. Expression of stc-1 mRNA in zebrafish embryos was increased in high-Ca(2+) environments but decreased by acidic and ion-deficient treatments while overexpression of stc-1 impaired the hypotonic acclimation by decreasing whole body Ca(2+), Na(+), and Cl(-) contents and H(+) secretion ability. Injection of STC-1 mRNA also down-regulated mRNA expressions of epithelial Ca(2+) channel, H(+)-ATPase, and Na(+)-Cl(-) cotransporter, suggesting the roles of STC-1 in regulation of ions other than Ca(2+). Knockdown of STC-1 caused an increase in ionocyte progenitors (foxi3a as the marker) and mature ionocytes (ion transporters as the markers), but did not affect epithelium stem cells (p63 as the marker) in the embryonic skin. Overexpression of STC-1 had the corresponding opposite effect on ionocyte progenitors, mature ionocytes in the embryonic skin. Taken together, STC-1 negatively regulates the number of ionocytes to reduce ionocyte functions. This process is important for body fluid ionic homeostasis, which is achieved by the regulation of ion transport functions in ionocytes. The present findings provide new insights into the broader functions of STC-1, a hypocalcemic hormone.

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.

Na+/K+-ATPase and vacuolar-type H+-ATPase in the gills of the aquatic air-breathing fish Trichogaster microlepis in response to salinity variation.

The aquatic air-breathing fish, Trichogaster microlepis, can be found in fresh water and estuaries. We further evaluated the changes in two important osmoregulatory enzymes, Na(+)/K(+)-ATPase (NKA) and vacuolar-type H(+)-ATPase (VHA), in the gills when fish were subjected to deionized water (DW), fresh water (FW), and salinated brackish water (salinity of 10 g/L). Fish were sampled only 4 days after experimental transfer. The mortality, plasma osmolality, and Na(+) concentration were higher in 10 g/L acclimated fish, while their muscle water content decreased with elevated external salinity. The highest NKA protein abundance was found in the fish gills in 10 g/L, and NKA activity was highest in the DW and 10 g/L acclimated fish. The VHA protein levels were highest in 10 g/L, and VHA activity was highest in the DW treatment. From immunohistochemical results, we found three different cell populations: (1) NKA-immunoreactive (NKA-IR) cells, (2) both NKA-IR and HA-IR cells, and (3) HA-IR cells. NKA-IR cells in the lamellar and interlamellar regions significantly increased in DW and 10 g/L treatments. Only HA-IR cells in the lamellar region were significantly increased in DW. In the interlamellar region, there was no difference in the number of HA-IR cells among the three treated. From these results, T. microlepis exhibited osmoregulatory ability in DW and 10 g/L treatments. The cell types involved in ionic regulation were also examined with immunofluorescence staining; three ionocyte types were found which were similar to the zebrafish model.