Ionocyte Differentiation Research Articles

Mineralocorticoid Receptor Mediates Cortisol Regulation of Ionocyte Development in Tilapia (Oreochromis mossambicus)

Cortisol is the predominant corticosteroid in ray-finned fish since it does not possess the aldosterone synthase necessary to produce specific mineralocorticoids. Cortisol is traditionally believed to function as a fish mineralocorticoid. However, the effects of cortisol are mediated through corticosteroid receptors in other vertebrates, and there is an ongoing debate about whether cortisol acts through the glucocorticoid receptor (GR) or the mineralocorticoid receptor (MR) in teleosts. To investigate this issue, we conducted a study using euryhaline Mozambique tilapia (Oreochromis mossambicus) as the experimental species. The experiment was designed to investigate the effect of cortisol on ionocyte development at both the cellular and gene expression levels in tilapia. We administered exogenous cortisol and receptor antagonists, used immunohistochemistry to quantify ionocyte numbers, and performed real-time PCR to assess the expression of the differentiation factor tumor protein 63 (P63) mRNA, an epidermal stem cell marker. We observed that cortisol increased the number of Na+-K+-ATPase (NKA)-immunoactive ionocytes (increased by 1.6-fold) and promoted the gene expression of P63 mRNA (increased by 1.4-fold). Furthermore, we found that the addition of the mineralocorticoid receptor antagonist Spironolactone inhibited the increase in the number of ionocytes (decreased to the level of the control group) and suppressed the gene expression of P63 (similarly decreased to the level of the control group). We also provided evidence for gr, mr, and p63 localization in epidermal cells. At the transcript level, mr mRNA is ubiquitously expressed in gill sections and present in epidermal stem cells (cells labeled with p63), supporting the antagonism and functional assay results in larvae. Our results confirmed that cortisol stimulates ionocyte differentiation in tilapia through the MR, rather than the GR. Therefore, we provide a new direction for investigating the dual action of osmotic regulation and skin/gill epithelial development in tilapia, which could help resolve previously inconsistent and conflicting findings.

Sonic Hedgehog Signaling Is Essential for Pulmonary Ionocyte Specification in Human and Ferret Airway Epithelia.

Pulmonary ionocytes express high levels of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that is critical for hydration of the airways and mucociliary clearance. However, the cellular mechanisms that govern ionocyte specification and function remain unclear. We observed that increased abundance of ionocytes in cystic fibrosis (CF) airway epithelium was associated with enhanced expression of Sonic Hedgehog (SHH) effectors. In this study, we evaluated whether the SHH pathway directly impacts ionocyte differentiation and CFTR function in airway epithelia. Pharmacological HPI1-mediated inhibition of SHH signaling component GLI1 significantly impaired human basal cell specification of ionocytes and ciliated cells but significantly enhanced specification of secretory cells. By contrast, activation of the SHH pathway effector smoothened (SMO) with the chemical agonist SAG significantly enhanced ionocyte specification. The abundance of CFTR+ BSND+ ionocytes under these conditions had a direct relationship with CFTR-mediated currents in differentiated air-liquid interface (ALI) airway cultures. These findings were corroborated in ferret ALI airway cultures generated from basal cells in which the genes encoding the SHH receptor PTCH1 or its intracellular effector SMO were genetically ablated using CRISPR-Cas9, causing aberrant activation or suppression of SHH signaling, respectively. These findings demonstrate that SHH signaling is directly involved in airway basal cell specification of CFTR-expressing pulmonary ionocytes and is likely responsible for enhanced ionocyte abundance in the CF proximal airways. Pharmacologic approaches to enhance ionocyte and reduce secretory cell specification after CFTR gene editing of basal cells may have utility in the treatment of CF.

Vitamin D regulates ion regulation by affecting the ionocyte differentiation in zebrafish (Danio rerio) larvae

Freshwater teleosts frequently face the stress of varied ion and pH levels; therefore, they have developed related defense mechanisms to maintain the homeostasis of body-fluid ion and acid-base balance. The different subtypes of ionocytes expressed in the branchial epithelium of adult fish or the skin of larvae are the major sites for fish ion regulation. 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the bioactive form of vitamin D, is a steroid hormone that is involved in the regulation of Ca2+ uptake and acid secretion in teleosts. Our results revealed that 1α,25(OH)2D3 levels were not changed in zebrafish larvae upon exposure to low Na+ freshwater compared to normal freshwater. In contrast, 1α,25(OH)2D3 levels were substantially higher in fish exposed to acidic and low Ca2+ freshwater than in those exposed to normal freshwater. Some hormones regulate ion regulation and acid secretion by modulating ionocyte differentiation and/or proliferation in teleosts; however, the role of vitamin D in this process is unclear. Zebrafish larvae were used as a model in the present study to explore the effect of vitamin D on ionocyte proliferation and/or differentiation. The present study indicated that 1α,25(OH)2D3 treatment increased the number of foxi3a-positive cells, ionocyte progenitors, and mature ionocytes. However, the number of P63-positive epidermal stem cells did not change in the zebrafish larvae treated with 1α,25(OH)2D3. These results revealed that vitamin D exerts a positive effect on the number of ionocytes by increasing the differentiation of ionocytes. Increased ionocyte differentiation by vitamin D is suggested to elevate the capacity of ion regulation and acid secretion in zebrafish to cope with external stress. The present findings indicate the role of vitamin D in the regulation of ionocyte differentiation and provide new insights into the mechanisms of stress adaptation of fish.

Differential Branchial Response of Low Salinity Challenge Induced Prolactin in Active and Passive Coping Style Olive Flounder.

Stress coping styles are very common in fish, and investigations into this area can greatly improve fish welfare and promote the sustainable development of aquaculture. Although most studies have focused on the behavioral and physiological differences of these fishes, the endocrine response of different coping styles fish when undergoing salinity challenge is still unclear. We examined the physiological response in olive flounder with active coping (AC) style and passive coping (PC) style after transferred from seawater (SW) to freshwater for 0, 2, 5, 8, and 14 days. The results showed that: 1) the plasma prolactin level of FW-acclimated AC flounder was substantially higher than that of FW-acclimated PC flounder at 5, 8, and 14 days, and the branchial gene expression of prolactin receptor (PRLR) in AC flounder was slightly higher than PC flounder after transfer. While there was no remarkable difference observed in cortisol (COR) levels between AC and PC flounder. After transfer, glucocorticoid receptor (GR) expression in AC flounder was significantly higher compared with PC flounder at 8 days. 2) Branchial NKA-IR ionocytes numbers were reduced in PC flounder after transfer, while ionocytes number remain stable in AC flounder. 3) The branchial stem cell transcription factor foxi1 gene expression of AC flounder was significantly higher than PC flounder at 2, 5, and 14 days after transfer, while branchial stem cell transcription factor p63 gene expression of FW-acclimated AC flounder was only substantially higher than that of PC flounder at 5 days. 4) As an apoptosis upstream initiator, the branchial gene expression of caspase-9 in PC flounder was considerably higher than in AC flounder after transfer at 8 days. This study revealed that olive flounder with active and passive coping styles have different endocrine coping strategies after facing the low-salinity challenge. AC flounder adopt an active endocrine strategy by increasing ionocyte differentiation and prolactin secretion significantly. In contrast, PC flounder employ a passive strategy of reducing ionocytes differentiation and retaining prolactin content at a low level to reduce branchial ionocytes number.

Atlantic salmon (Salmo salar) exposed to different preparatory photoperiods during smoltification show varying responses in gill Na+/K+-ATPase, salinity-specific mRNA transcription and ionocyte differentiation

Control of the parr-smolt transformation (or smoltification) is crucial for the husbandry and successful seawater (SW) transfer of Atlantic salmon (Salmo salar) reared in freshwater (FW) hatcheries. Photoperiod is an important environmental signal that initiates the complex physiological, morphological and behavioural changes that coincide with marine migration. While the use of long-day photoperiods to initiate smoltification has been well studied, this study investigated how three preparatory photoperiods in FW (LD 08:16, LD 12:12, LD 16:08) preceding exposure to 24-h light (LD 24:0) may influence or enhance smolt performance and growth post-SW transfer. After the photoperiod treatment phase (8 weeks), all groups were exposed to LD 24:0 for 8 weeks (FW) and then transferred to SW for a further 8 weeks. Exposure to LD 16:08 induced rapid development of smolt-related characteristics such as increased gill NKA activity, gill NKAα1b mRNA, and plasma cortisol, and decreased gill NKAα1a mRNA levels and condition factor through the 8-week treatment phase. Subsequent exposure to a LD 24:0 photoperiod resulted in a partial reversal of several of these characteristics, suggesting these fish went through a partial desmoltification. Exposure to LD 12:12 for 8 weeks prior to LD 24:0 elicited an intermediary response in smoltification attributes compared to LD 16:08 and LD 08:16. The LD 12:12 group adapted to SW and showed no negative effects on growth or physiological responses after transfer to SW. Exposure to a shortened photoperiod (LD 08:16) did not elicit any smoltification-related changes prior to LD 24:0, however, exposure to LD 24:0 increased gill NKA activity, plasma cortisol, changes in NKAα1a and NKAα1b mRNA, and the ratio of NKAα1b: NKAα1a. These results were confirmed by the expected changes in NKAα1a and NKAα 1b-positive immuno-reactive gill ionocytes. In summary, after exposure to LD 24:0 fish in the LD 08:16 group showed similar levels of change to those of the LD 16:08 group during the initial FW phase (prior to exposure to LD 24:0). After SW transfer, all groups were able to upregulate SW-specific NKAα1b mRNA and acclimate to SW, even though no increase in cortisol was evident. By the end of the study, there was no difference in SW growth among the groups. Overall, our data indicate that LD 16:08 advanced hypoosmoregulatory characteristics prior to LD 24:0 exposure. In addition, the physiological and molecular indicators measured in this group suggest that fish could have been transferred to SW immediately after 8 weeks in LD 16:08, with no added benefit of successive exposure to LD 24:0, which is typically used by industry to induce smoltification.

Evidence for two distinct waves of epidermal ionocyte differentiation during medaka embryonic development.

The fish epidermis contains specific cells, or ionocytes, that are specialized in ion transport and contribute to the osmoregulatory function. Besides the zebrafish model, the medaka (Oryzias latipes) has recently emerged as an important model for osmoregulation studies because it possesses a particularly high adaptability to salinity changes. However, hindering the progress of research on embryonic ionocytes is the lack of a comprehensive view of their developmental dynamic. Using EdU integrations and the foxi3 and NKA markers, we characterized the proliferating progenitors of ionocytes (here called ionoblastes) and we quantified them, along with ionocytes, during embryogenesis. While progenitors of the vitellin zone promptly differentiate in a synchronous manner, progenitors of the lateral zone differentiate progressively and asynchronously. Furthermore, we evidenced that nhe3 is expressed in differentiated ionocytes of both zones, whereas ecac, ncc, and gcm2 are strictly specific of the lateral zone. We also evidenced that the two zones are differentially regulated in distilled water and seawater. Our data led us to propose a model timeline, which provides evidence for the expansion of two successive and distinct populations of ionocytes. This model opens the way for new studies related to epidermal development, plasticity and osmoregulation ontogeny.

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.

Expression of Ol-foxi3 and Na +/K +-ATPase in ionocytes during the development of euryhaline medaka ( Oryzias latipes) embryos

Osmoregulation is a vital function that is essential to all vertebrates. Ionocytes are epithelial cells responsible for this function and have been extensively studied in adult teleost fish gills. The euryhaline medaka ( Oryzias latipes) has recently emerged as an investigative model because of its ability to acclimatize easily to water presenting various salinities. However, no studies to date have focused on the development of ionocytes in medaka embryos. We first analyzed the distribution of ionocytes in the skin and gills during development, using a specific marker of differentiated ionocytes (the Na +/K +-ATPase pump, or NKA). Strikingly, we were able to identify two ionocyte domains on the yolk surface ectoderm, that we named the Vitellin Zone (VZ) and the Lateral Zone (LZ). In zebrafish, ionocyte differentiation has been shown to be controlled by two forkhead-box genes, foxi3a and foxi3b. We cloned the medaka foxi3 ortholog which appeared to be highly similar to foxi3b. Whole-mount in situ hybridizations performed on medaka embryos revealed that Ol-foxi3 is expressed in differentiated ionocytes of the pharyngeal endoderm, the branchial arches and the yolk epidermis, as well as in epibranchial placode territories. We further focused on the expression patterns of the yolk epidermis and compared the expression of Ol-foxi3 with that of the non-neural progenitor marker p63. We evidenced that Ol-foxi3 is expressed in progenitor cells which are first of all located uniformly in the VZ and then transitorily clustered in the LZ. Taken together, these data contribute to a clearer understanding of osmoregulatory tissue ontogenesis in euryhaline fish.

Zebrafish grainyhead-like1 is a common marker of different non-keratinocyte epidermal cell lineages, which segregate from each other in a Foxi3-dependent manner

Grainyhead/CP2 transcription factor family members are widely conserved among the animal kingdom and have been implicated in different developmental processes. Thus far, nothing has been known about their roles in zebrafish. Here we identify seven zebrafish grainyhead-like (grhl) / cp2 genes, with focus on grhl1, which is expressed in the periderm and in epidermal ionocyte progenitors, but downregulated when ionocytes differentiate. In addition, expression was detected in other "non-keratinocyte" cell types of the epidermis, such as pvalb8-expressing cells, which according to our lineage tracing experiments are derived from the same pool of progenitor cells like keratinocytes and ionocytes. Antisense morpholino oligonucleotide-based loss-of-function analysis revealed that grhl1 is dispensable for the development and function of all investigated epidermal cell types, but required as a negative regulator of its own transcription during ionocyte differentiation. Knockdown of the transcription factor Foxi3a, which is expressed in a subset of the grhl1 population, caused a loss of ionocytes and a corresponding increase in the number of pvalb8-expressing cells, while leaving the number of grhl1-positive cells unaltered. We propose that grhl1 is a novel common marker of all or most "non-keratinocyte" epidermal progenitors, and that the sub-functionalisation of these cells is regulated by differential positive and negative effects of Foxi3 factors.

Changing expression patterns: focus on “The transcription factor, glial cell missing 2, is involved in differentiation and functional regulation of H+-ATPase-rich cells in zebrafish (Danio rerio)”

euryhaline species of fish have a remarkable osmo- and ionoregulatory capacity. Some euryhaline species like the Mozambique tilapia can directly be transferred from freshwater to seawater and vice versa, i.e., osmolarity of the external fluid changes by ∼1,000 mOsm. Many others can tolerate this

Foxi3 transcription factors and Notch signaling control the formation of skin ionocytes from epidermal precursors of the zebrafish embryo

Ionocytes are specialized epithelial cell types involved in the maintenance of osmotic homeostasis. In amniotes, they are present in the renal system, while in water-living embryos of lower vertebrates additional ionocytes are found in the skin. Thus far, relatively little has been known about the mechanisms of ionocyte development. Here we demonstrate that skin ionocytes of zebrafish embryos derive from the same precursor cells as keratinocytes. Carrying out various combinations of gain- and loss-of-function studies, we show that the segregation of ionocytes from the epidermal epithelium is governed by an interplay between Notch signaling and two Forkhead-box transcription factors, Foxi3a and Foxi3b. The two foxi3 genes are expressed in ionocyte precursors and are required both for ionocyte-specific expression of the Notch ligand Jagged2a, and for ionocyte differentiation, characterized by the production of particular ATPases. Ionocytic Notch ligands, in turn, signal to neighboring cells, where activated Notch1 leads to a repression of foxi3 expression, allowing those cells to become keratinocytes. A model for ionocyte versus keratinocyte development will be presented, postulating additional thus far unidentified pro-ionocyte factors.

A Positive Regulatory Loop between foxi3a and foxi3b Is Essential for Specification and Differentiation of Zebrafish Epidermal Ionocytes

BackgroundEpidermal ionocytes play essential roles in the transepithelial transportation of ions, water, and acid-base balance in fish embryos before their branchial counterparts are fully functional. However, the mechanism controlling epidermal ionocyte specification and differentiation remains unknown.Methodology/Principal FindingsIn zebrafish, we demonstrated that Delta-Notch-mediated lateral inhibition plays a vital role in singling out epidermal ionocyte progenitors from epidermal stem cells. The entire epidermal ionocyte domain of genetic mutants and morphants, which failed to transmit the DeltaC-Notch1a/Notch3 signal from sending cells (epidermal ionocytes) to receiving cells (epidermal stem cells), differentiates into epidermal ionocytes. The low Notch activity in epidermal ionocyte progenitors is permissive for activating winged helix/forkhead box transcription factors of foxi3a and foxi3b. Through gain- and loss-of-function assays, we show that the foxi3a-foxi3b regulatory loop functions as a master regulator to mediate a dual role of specifying epidermal ionocyte progenitors as well as of subsequently promoting differentiation of Na+,K+-ATPase-rich cells and H+-ATPase-rich cells in a concentration-dependent manner.Conclusions/SignificanceThis study provides a framework to show the molecular mechanism controlling epidermal ionocyte specification and differentiation in a low vertebrate for the first time. We propose that the positive regulatory loop between foxi3a and foxi3b not only drives early ionocyte differentiation but also prevents the complete blockage of ionocyte differentiation when the master regulator of foxi3 function is unilaterally compromised.