Pulmonary Ionocytes Regulate Airway Surface Liquid pH in Primary Human Bronchial Epithelial Cells.

Rationale: Pulmonary ionocytes are a newly discovered airway epithelial cell type proposed to be a major contributor to cystic fibrosis (CF) lung disease based on observations they express the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel at a higher level than any other cell type in the airway epithelia. Moreover, genetically manipulated experimental models that lack ionocytes develop NaCl transport abnormalities and airway surface liquid (ASL) dehydration consistent with CF. However, no direct evidence indicates ionocytes engage in NaCl transport or contribute to ASL formation, questioning the relevance of ionocytes to CF lung disease. Objectives: To determine the ion transport properties of pulmonary ionocytes and club cells in genetically intact healthy and CF airway epithelia. Methods: We measured ion transport at the single-cell level using a self-referencing ion-selective microelectrode technique in primary human bronchial epithelial cell culture. Measurements and Main Results: cAMP-stimulated non-CF ionocytes do not secrete Na+ or Cl- into the ASL, but rather modulate its pH by secreting bicarbonate via CFTR-linked Cl-/bicarbonate exchange. Non-CF club cells secrete Na+ and Cl- to the lumen side after cAMP stimulation. CF ionocytes and club cells do not transport ions in response to cAMP stimulation, but incubation with CFTR modulators elexacaftor/tezacaftor/ivacaftor restores transport properties. Conclusions: We conclude that ionocytes do not contribute to ASL formation but regulate ASL pH. Club cells secrete the bulk of airway fluid. In CF, abnormal ionocyte and club cell function results in acidic and dehydrated ASL, causing reduced antimicrobial properties and mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

In vitro platform to model the function of ionocytes in the human airway epithelium.

BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.

Exposure to Total Suspended Solids (TSS) Impacts Gill Structure and Function in Adult Zebrafish.

Total suspended solids (TSS) are a major contributor of anthropogenic impacts to aquatic systems. TSS exposure have been shown to affect the function of gills, but the mode of action is unclear. Zebrafish (Danio rerio) is emerging as an excellent model for mechanistic toxicology, and as there are no baseline studies on TSS effects in zebrafish gills, we tested the hypothesis that environmental concentrations of TSS damages gill structure and function in this species. Adult zebrafish were exposed to either 0, 10, 100, 500, 1000, or 2000 mg/L TSS for 4 days to assess the gill morphology. The minimal concentration that affected the gill structure was further tested for the distribution of key ion transporters, including Na+/K+- ATPase (NKA) and vacuolar-type H+-ATPase (VHA), using confocal microscopy. Our results reveal that TSS concentration as low as 100 mg/L alters the morphology of gills, including greater filament thickness, lamellae thickness, and epithelial lifting. This was also associated with a reduction in NKA immunoreactive (IR) cell count and intensity in the 100 mg/L TSS group, while there was neither a change in the VHA-IR cell count or expression nor the transcript abundance of atp6v1a and atp1a1a4 in the gills. Markers of stress response in these animals, including levels of cortisol, glucose, lactate, and glycogen were not altered after 4 days of TSS exposure. Overall, environmentally relevant concentrations of TSS can damage the gill structure and function in zebrafish and has the potential to enhance the toxicity of contaminants acting via the gills.

Physiological and immune profiling of tilapia Oreochromis niloticus gills by high-throughput single-cell transcriptome sequencing.

The physiological and immune functions of fish gills are largely recognized, but their following functional heterogeneity at the single cell scale has been rarely reported. Here, we performed single cell RNA sequencing (scRNA-seq) on the gills of tilapia fish Oreochromis niloticus. We identified a total of 12 cell populations and analyzed their functional heterogeneity. To investigate the physiological function of O. niloticus gills, expression patterns of genes encoding ion transporters were selected from the identified H+-ATPase-rich cells (HR cells), Na+/K+-ATPase-rich cells (NaR cells), and pavement cells. Specific enrichment of ca4a, slc9a1a, and LOC100692482 in the HR cells of O. niloticus gills explained their functions in acid-base regulation. Genes encoding Ca2+ transporters, including atp2b1, LOC100696627, and LOC 100706765, were specifically expressed in the NaR cells. Pavement cells were presumably the main sites responsible for ammonia and urea transports in O. niloticus gills with specific enrichment of Rhbg and LOC100693008, respectively. The expression patterns of the four immune cell subtypes varied greatly, with B cells being enriched with the most immune-related GO terms. KEGG enrichment analysis showed that MAPK signaling pathway was the most enriched pathway among the four types of immune cells in O. niloticus gills. Our results are important in understanding the physiological and immune responses of fish gills at the cellular resolution.

Acquired CFTR dysfunction and dense distribution of ionocytes in nasal mucosa of children with CRS.

BACKGROUND: Ionocytes are rare cells in airway epithelium characterized by a high expression of CFTR. OBJECTIVES: To investigate the morphology and distribution of ionocytes and the function of CFTR in the nasal mucosal epithelium of children. METHODS: The exfoliated cells of nasal mucosa from 101 children were detected using flow cytometry to analyze the number of ionocytes and CFTR and the difference of CFTR function. Nasal mucosa and polyps were collected from 10 children with CRSwNP. The RNAscope of FOXI1 and CFTR was detected in pathological paraffin sections. The expression and distribution of ionocytes and CFTR in nasal mucosa and polyp epithelium were observed. RESULTS: In CRS patients, the number of ionocytes in the nasal epithelium was lower and the number of ionocytes that did not express CFTR was higher, and the function of CFTR was also decreased. The expression of CFTR in the nasal mucosa of CRS showed the characteristics of local dense distribution and increased as the inflammation expanded. The ionocytes were "tadpole-shaped" in the epithelium and gathered in the area of high CFTR expression, the intracellular CFTR was expanded in clusters. Ionocytes that did not express CFTR was more common in the nasal polyps. CONCLUSIONS: The number of ionocytes and the function of CFTR in nasal mucosa of CRS patients decreased. With the expansion of inflammation, CFTR and ionocytes showed more obvious dense distribution. Some ionocytes lost the expression of CFTR and did not show the "tadpole" shape, which may be related to the occurrence of polyps.

Rapid blood acid-base regulation by European sea bass (Dicentrarchus labrax) in response to sudden exposure to high environmental CO2.

Fish in coastal ecosystems can be exposed to acute variations in CO2 of between 0.2 and 1 kPa CO2 (2000-10,000 µatm). Coping with this environmental challenge will depend on the ability to rapidly compensate for the internal acid-base disturbance caused by sudden exposure to high environmental CO2 (blood and tissue acidosis); however, studies about the speed of acid-base regulatory responses in marine fish are scarce. We observed that upon sudden exposure to ∼1 kPa CO2, European sea bass (Dicentrarchus labrax) completely regulate erythrocyte intracellular pH within ∼40 min, thus restoring haemoglobin-O2 affinity to pre-exposure levels. Moreover, blood pH returned to normal levels within ∼2 h, which is one of the fastest acid-base recoveries documented in any fish. This was achieved via a large upregulation of net acid excretion and accumulation of HCO3- in blood, which increased from ∼4 to ∼22 mmol l-1. While the abundance and intracellular localisation of gill Na+/K+-ATPase (NKA) and Na+/H+ exchanger 3 (NHE3) remained unchanged, the apical surface area of acid-excreting gill ionocytes doubled. This constitutes a novel mechanism for rapidly increasing acid excretion during sudden blood acidosis. Rapid acid-base regulation was completely prevented when the same high CO2 exposure occurred in seawater with experimentally reduced HCO3- and pH, probably because reduced environmental pH inhibited gill H+ excretion via NHE3. The rapid and robust acid-base regulatory responses identified will enable European sea bass to maintain physiological performance during large and sudden CO2 fluctuations that naturally occur in coastal environments.

Secretory Cells Dominate Airway CFTR Expression and Function in Human Airway Superficial Epithelia.

Rationale: Identification of the specific cell types expressing CFTR (cystic fibrosis [CF] transmembrane conductance regulator) is required for precision medicine therapies for CF. However, a full characterization of CFTR expression in normal human airway epithelia is missing. Objectives: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single-cell RNA (scRNA) sequencing (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. scRNA in situ hybridization (ISH) and single-cell qRT-PCR were performed for validation. In vitro culture systems correlated CFTR function with cell types. Lentiviruses were used for cell type-specific transduction of wild-type CFTR in CF cells. Measurements and Main Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and, particularly, small airway superficial epithelia, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, whereas the expression in ciliated cells was infrequent and low. scRNA ISH and single-cell qRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to those of normal control subjects. CFTR mediated Cl- secretion in cultures tracked secretory cell, but not ionocyte, densities. Furthermore, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells and not with ionocytes. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not in ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.

Rapid embryonic development supports the early onset of gill functions in two coral reef damselfishes.

The gill is one of the most important organs for growth and survival of fishes. Early life stages in coral reef fishes often exhibit extreme physiological and demographic characteristics that are linked to well-established respiratory and ionoregulatory processes. However, gill development and function in coral reef fishes is not well understood. Therefore, we investigated gill morphology, oxygen uptake and ionoregulatory systems throughout embryogenesis in two coral reef damselfishes, Acanthochromis polyacanthus and Amphiprion melanopus (Pomacentridae). In both species, we found key gill structures to develop rapidly early in the embryonic phase. Ionoregulatory cells appear on gill filaments 3-4 days post-fertilization and increase in density, whilst disappearing or shrinking in cutaneous locations. Primary respiratory tissue (lamellae) appears 5-7 days post-fertilization, coinciding with a peak in oxygen uptake rates of the developing embryos. Oxygen uptake was unaffected by phenylhydrazine across all ages (pre-hatching), indicating that haemoglobin is not yet required for oxygen uptake. This suggests that gills have limited contribution to respiratory functions during embryonic development, at least until hatching. Rapid gill development in damselfishes, when compared with that in most previously investigated fishes, may reflect preparations for a high-performance, challenging lifestyle on tropical reefs, but may also make reef fishes more vulnerable to anthropogenic stressors.

Effects of seawater acclimation on two Na+/K+-ATPase α-subunit isoforms in the gills of the marble goby, Oxyeleotris marmorata.

The marble goby, Oxyeleotris marmorata, is a freshwater teleost, but can acclimate progressively to survive in seawater (salinity 30). As an obligatory air-breather, it can also survive long periods of emersion. Two isoforms of Na+/K+-ATPase (nka) α-subunit, nkaα1 and nkaα3, but not nkaα2, had been cloned from the gills of O. marmorata. The cDNA sequence of nkaα1 consisted of 3069 nucleotides, coding for 1023 amino acids (112.5 kDa), whereas nkaα3 consisted of 2976 nucleotides, coding for 992 amino acids (109.5 kDa). As only one form of branchial Nkaα1 was identified using molecular cloning in this study, O. marmorata lacks specific freshwater- and seawater-type Nkaα isoforms as demonstrated by some other euryhaline fish species. The nkaα1 transcript level was about 2.5-fold higher than that of nkaα3 in the gills of freshwater O. marmorata. During exposure to seawater, the branchial transcript level of nkaα1 increased significantly on day 1 (~3.3-fold) and day 6 (~2.6-fold). By contrast, the branchial transcript level of nkaα3 increased significantly on day 1 (~2.6-fold), but not on day 6, of seawater exposure. Six days of exposure to seawater also led to significant increases in protein abundances of Nkaα1 (~6.9-fold) and Nkaα3 (~2.8-fold) in the gills of O. marmorata. Hence, the mRNA and protein expressions of both nkaα1/Nkaα1 and nkaα3/Nkaα3 were up-regulated in O. marmorata during seawater acclimation. This could explain why Vmax increases but Km for Na+ and K+ remain unchanged in Nka extracted from the gills of O. marmorata acclimated to seawater as reported previously.

Enhanced expression of ncc1 and clc2c in the kidney and urinary bladder accompanies freshwater acclimation in Mozambique tilapia.

Euryhaline fishes maintain hydromineral balance in a broad range of environmental salinities via the activities of multiple osmoregulatory organs, namely the gill, gastrointestinal tract, skin, kidney, and urinary bladder. Teleosts residing in freshwater (FW) environments are faced with the diffusive loss of ions and the osmotic gain of water, and, therefore, the kidney and urinary bladder reabsorb Na+ and Cl- to support the production of dilute urine. Nonetheless, the regulated pathways for Na+ and Cl- transport by euryhaline fishes, especially in the urinary bladder, have not been fully resolved. Here, we first investigated the ultrastructure of epithelial cells within the urinary bladder of FW-acclimated Mozambique tilapia (Oreochromis mossambicus) by electron microscopy. We then investigated whether tilapia employ Na+/Cl- cotransporter 1 (Ncc1) and Clc family Cl- channel 2c (Clc2c) for the reabsorption of Na+ and Cl- by the kidney and urinary bladder. We hypothesized that levels of their associated gene transcripts vary inversely with environmental salinity. In whole kidney and urinary bladder homogenates, ncc1 and clc2c mRNA levels were markedly higher in steady-state FW- versus SW (seawater)-acclimated tilapia. Following transfer from SW to FW, ncc1 and clc2c in both the kidney and urinary bladder were elevated within 48 h. A concomitant increase in branchial ncc2, and decreases in Na+/K+/2Cl-cotransporter 1a (nkcc1a) and cystic fibrosis transmembrane regulator 1 (cftr1) levels indicated a transition from Na+ and Cl- secretion to absorption by the gills in parallel with the identified renal and urinary bladder responses to FW transfer. Our findings suggest that Ncc1 and Clc2c contribute to the functional plasticity of the kidney and urinary bladder in tilapia.

The osmorespiratory compromise in the fish gill.

August Krogh made fundamental discoveries about both respiratory gas exchange and osmo/iono-regulation in fish gills. Dave Randall and co-workers identified a tradeoff between these two functions such that high functional surface area and low diffusion distance would favour O2 uptake (e.g. exercise, hypoxia), whereas low functional surface area and high diffusion distance would favour osmo/iono-regulation (rest, normoxia). Today we call this concept the "osmorespiratory compromise" and realize that it is much more complex than originally envisaged. There are at least 6 mechanisms by which fish can change functional branchial area and diffusion distance. Three involve reorganizing blood flow pathways: (i) flow redistribution within the secondary (respiratory) lamellae; (ii) flow shunting between "respiratory" and "ionoregulatory" pathways in the filament; (iii) opening up more distal lamellae on the filament and closing non-respiratory pathways. Three more involve "reversible gill remodeling": (iv) proliferation of the interlamellar gill cell mass (ILCM); (v) proliferation of ionocytes up the sides of the lamellae; (vi) covering over the apical exposure of ionocytes by extension of pavement cells. In ways that remain incompletely understood, these mechanisms allow dynamic regulation of the osmorespiratory compromise, such that ion and water fluxes can be decoupled from O2 uptake during continuous exercise. Furthermore, hypoxia-tolerant species can reduce branchial ion and water fluxes below normoxic levels despite hyperventilating during hypoxia. In marine fish, the osmorespiratory conflict is intensified by the greater ionic and osmotic gradients from seawater to blood, but underlying mechanisms remain poorly understood.

Effects of salinity on gills' chloride cells, stress indices, and gene expression of Asian seabass (Lates calcarifer, Bloch, 1790).

A 2-week research was carried out to assess water salinity (WS) effects including 0, 15, 35, and 50‰ on osmoregulatory mechanisms and stress indices in Asian sea bass (34.4 g) juveniles. Except for fish reared at 50‰, in the other treatments, it gradually decreased to the prescribed WS during a 10-day period (- 5‰ a day). After a 10-day acclimation period, fish were reared at the prescribed WS for 2 weeks. Fish reared at 15 and 35‰ had higher chloride cell (CC) counts in the interlamellar region. The number of CC in the interlamellar region elevated with increment of WS up to 35‰, but they were pronouncedly reduced in 50‰ group. The diameter of CC in the interlamellar region was not affected by WS. The smallest nucleus diameter of CC in the interlamellar region was observed in fish reared at 15‰ (P < 0.05). The largest and the smallest amounts of serum aspartate aminotransferase were observed in fish reared at freshwater and 15‰, respectively. Fish reared at 35‰ had the highest serum sodium and potassium contents. Serum chloride content and total osmolality increased with increment of WS (P < 0.05). Serum cortisol and glucose contents gradually increased with elevation of WS up to 35‰; then, their contents remarkably decreased. The relative expression of insulin like growth factor-1 in the liver of fish reared at 35‰ was strikingly higher than that in the other groups. The relative expression of HSP70 gene in fresh water group was pronouncedly elevated compared to other treatments. The relative expression of interleukin-1ß in 15 and 35‰ groups was higher than that in the other groups; however, the relative expression of lysozyme gene in the liver of fish reared at fresh water was pronouncedly lower than that in the other treatments. The results of this study suggested rearing L. calcarifer at 15‰ closer to the isosmotic point and better provide its welfare.

Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes.

European sea bass Dicentrarchus labrax undertake seasonal migrations to estuaries and lagoons that are characterized by fluctuations in environmental conditions. Their ability to cope with these unstable habitats is undeniable, but it is still not clear how and to what extent salinity acclimation mechanisms are affected at temperatures higher than in the sea. In this study, juvenile sea bass were pre-acclimated to seawater (SW) at 18°C (temperate) or 24°C (warm) for 2weeks and then transferred to fresh water (FW) or SW at the respective temperature. Transfer to FW for two weeks resulted in decreased blood osmolalities and plasma Cl- at both temperatures. In FW warm conditions, plasma Na+ was ~15% lower and Cl- was ~32% higher than in the temperate-water group. Branchial Na+/K+-ATPase (NKA) activity measured at the acclimation temperature (Vapparent) did not change according to the conditions. Branchial Na+/K+-ATPase activity measured at 37°C (Vmax) was lower in warm conditions and increased in FW compared to SW conditions whatever the considered temperature. Mitochondrion-rich cell (MRC) density increased in FW, notably due to the appearance of lamellar MRCs, but this increase was less pronounced in warm conditions where MRC's size was lower. In SW warm conditions, pavement cell apical microridges are less developed than in other conditions. Overall gill morphometrical parameters (filament thickness, lamellar length and width) differ between fish that have been pre-acclimated to different temperatures. This study shows that a thermal change affects gill plasticity affecting whole-organism ion balance two weeks after salinity transfer.

Assessing the role of the acid-sensing ion channel ASIC4b in sodium uptake by larval zebrafish.

Na+ uptake in larval zebrafish (Danio rerio) is coordinated by three mechanisms: Na+/H+-exchanger 3b (NHE3b) expressed in H+-ATPase-rich (HR) cells, an unidentified Na+ channel coupled to electrogenic H+-ATPase expressed in HR cells, and Na+-Cl--cotransporter (NCC) expressed in NCC cells. Recently, acid-sensing ion channels (ASICs) were proposed to be the putative Na+ channel involved in H+-ATPase-mediated Na+ uptake in adult zebrafish and rainbow trout. In the present study, we hypothesized that ASICs also play this role in Na+ uptake in larval zebrafish. In support of this hypothesis, immunohistochemical analyses revealed that ASIC4b was expressed in HR cells on the yolk sac skin at 4 days post-fertilization (dpf). However, neither treatment with the ASIC-specific blocker 4,6-diamidino-2-phenylindole (DAPI) nor morpholino knockdown of ASIC4b reduced Na+ uptake in circumneutral conditions at 4 dpf. However, because ASIC4b knockdown led to significant increases in the mRNA expression of nhe3b and ncc and a significant increase in HR cell density, it is possible that Na+ influx was sustained by increased participation of non-ASIC4b pathways. Moreover, when fish were reared in acidic water (pH = 4), ASIC4b knockdown led to a stimulation of Na+ uptake at 3 and 4 dpf, results which also were inconsistent with an essential role for ASIC-mediated Na+ uptake, even under conditions known to constrain Na+ uptake via NHE3b. Thus, while ASIC4b clearly is expressed in HR cells, the current functional experiments cannot confirm its involvement in Na+ uptake in larval zebrafish.

Zebrafish as a Model System for Investigating the Compensatory Regulation of Ionic Balance during Metabolic Acidosis.

Zebrafish (Danio rerio) have become an important model for integrative physiological research. Zebrafish inhabit a hypo-osmotic environment; to maintain ionic and acid-base homeostasis, they must actively take up ions and secrete acid to the water. The gills in the adult and the skin at larval stage are the primary sites of ionic regulation in zebrafish. The uptake of ions in zebrafish is mediated by specific ion transporting cells termed ionocytes. Similarly, in mammals, ion reabsorption and acid excretion occur in specific cell types in the terminal region of the renal tubules (distal convoluted tubule and collecting duct). Previous studies have suggested that functional regulation of several ion transporters/channels in the zebrafish ionocytes resembles that in the mammalian renal cells. Additionally, several mechanisms involved in regulating the epithelial ion transport during metabolic acidosis are found to be similar between zebrafish and mammals. In this article, we systemically review the similarities and differences in ionic regulation between zebrafish and mammals during metabolic acidosis. We summarize the available information on the regulation of epithelial ion transporters during acidosis, with a focus on epithelial Na⁺, Cl- and Ca2+ transporters in zebrafish ionocytes and mammalian renal cells. We also discuss the neuroendocrine responses to acid exposure, and their potential role in ionic compensation. Finally, we identify several knowledge gaps that would benefit from further study.

Development of osmoregulatory tissues in the Lake van fish (Alburnus tarichi) during larval development.

Lake Van is one of the largest alkaline lakes worldwide and Lake Van Fish (Alburnus tarichi Güldenstädt, 1814) is the only vertebrate species inhabiting it. Lake Van Fish is an anadromous species that migrates to the streams (salinity 0.02%, pH 8.42) flowing into Lake Van (salinity 0.22%, pH 9.8) during the spawning period (April-July). Following spawning, fish return to Lake Van while larvae remain in fresh water. This study examined the development of osmoregulatory organs and the distribution of ionocytes in Lake Van Fish larvae adapting to the highly alkaline water characterizing the lake. Ionocytes were marked immunohistochemically and observed in whole mounts with immunofluorescence staining using the Na+/K+ ATPase antibody. Ionocytes were first identified in the yolk sac membrane and skin, and then in the gills, digestive tract, and kidneys of larvae. The number of ionocytes on yolk sac membrane and skin decreased during larval development, indicating ionocytes on these tissues have a role in larvae osmoregulation. Larvae hatched from eggs in stream waters die when transferred to Lake Van water but survived in lake water diluted with deionized water. Thus, larvae need to go through certain alterations at the cellular and organ levels in order to adapt to the conditions of Lake Van water, indicating they do not enter this lake immediately after hatching.

The expression of VILL protein is hypoosmotic-dependent in the lamellar gill ionocytes of Otocephala teleost fish, Chanos chanos.

Milkfish, a species within the primitive teleost lineage Otocephala, can survive in water conditions ranging from hypo- to hyper-saline. This study explored the effects of environmental salinity on apical morphologies of ionocytes and the expression of villin homologs in the gills of milkfish acclimated to either seawater (SW) or fresh water (FW). Scanning electron microscopy revealed that the ionocytes in the gill filaments of SW and FW milkfish, respectively, cellular apical morphologies were hole-type and squint-type. The flat-type ionocytes were observed in the gill lamellae of FW milkfish. Furthermore, apical surfaces of some lamellar ionocytes exhibited microvilli. Villin 1 is a microvilli marker expressed in the epithelial cells of various vertebrates. In the phylogenetic tree of villin 1 homologs, primitive teleosts exhibit villin 1-like (VILL) and villin 1 proteins. Two mRNA sequences, villin 1 and VILL, were identified from the milkfish transcriptome by next generation sequencing. Low but constant expression of villin 1 (gene and protein) was observed in the gills for both SW and FW fish. VILL gene and protein expression levels in the gills were higher in FW fish, compared to SW fish. Double immunofluorescence staining demonstrated that VILL protein was present in some lamellar ionocytes of FW milkfish, but not in the filament ionocytes of either FW or SW milkfish. Taken together, these findings indicated that the VILL expression of ionocytes is hypoosmotic-dependent. The VILL might be involved in the formation of microvilli in the lamellar ionocytes for hyperosmoregulation of the milkfish.

Prolactin and cortisol mediate the maintenance of hyperosmoregulatory ionocytes in gills of Mozambique tilapia: Exploring with an improved gill incubation system.

Endocrine control of osmoregulation is essential for teleosts to adapt to various aquatic environments. Prolactin (PRL) is known as a fundamental endocrine factor for hyperosmoregulation in teleost fishes, acting on ionocytes in the gills to maintain ion concentrations of body fluid within narrow physiological ranges in freshwater conditions. Cortisol is also known as an osmoregulation-related steroid in teleosts; however, its precise function is still controversial. Here, we investigated more detailed effects of PRL and roles of cortisol on ionocytes of Mozambique tilapia (Oreochromis mossambicus) in freshwater, using an improved gill filament incubation system. This incubation system resulted in enhanced cell viability, as evaluated using the dead cell marker propidium iodide. PRL was shown to maintain the density of freshwater-type ionocytes in isolated gill filaments; this effect of PRL is not achieved by the activation of cell proliferation, but by the maintenance of existing ionocytes. Cortisol alone did not show any distinct effect on ionocyte density in isolated gill filaments. We also assessed effects of PRL and cortisol on relative mRNA levels of NCC2, NHE3, NKAa1a, and NKAa1b. PRL maintained relative NCC2 and NKAa1a mRNA abundance, and cortisol showed a stimulatory effect on relative NCC2 and NKAa1a mRNA levels in combination with PRL, though cortisol alone exerted no effect on these genes. An increase in NKAa1b mRNA abundance was detected in cortisol-treated groups. PRL treatment also maintained normal NCC2 localization at the apical membrane of the ionocytes. These results indicate that PRL maintains freshwater-type ionocytes, and that cortisol stimulates the function of ionocytes maintained by PRL.

Distribution and dynamics of branchial ionocytes in houndshark reared in full-strength and diluted seawater environments.

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranchs, however, the ionoregulatory function of the gills is still incompletely understood. Although two types of ionocytes, Na(+)/K(+)-ATPase (NKA)-rich (type-A) cell and vacuolar-type H(+)-ATPase (V-ATPase)-rich (type-B) cell, have been found in elasmobranch fishes, these cells were considered to function primarily in acid-base regulation. In the present study, we examined ion-transporting proteins expressed in ionocytes of Japanese-banded houndshark, Triakis scyllium, reared in full-strength seawater (SW) and transferred to diluted (30%) SW. In addition to the upregulation of NKA and Na(+)/H(+) exchanger type 3 (NHE3) mRNAs in the type-A ionocytes, we found that Na(+), Cl(-) cotransporter (NCC, Slc12a3) is expressed in a subpopulation of the type-B ionocytes, and that the expression level of NCC mRNA was enhanced in houndsharks transferred to a low-salinity environment. These results suggest that elasmobranch gill ionocytes contribute to NaCl uptake in addition to the already described function of acid-base regulation, and that NCC is most probably one of the key molecules for hyper-osmoregulatory function of elasmobranch gills. The existence of two types of ionocytes (NHE3- and NCC-expressing cells) that are responsible for NaCl absorption seems to be a common feature in both teleosts and elasmobranchs for adaptation to a low salinity environment. A possible driving mechanism for NCC in type-B ionocytes is discussed.

Prolactin 177, prolactin 188, and extracellular osmolality independently regulate the gene expression of ion transport effectors in gill of Mozambique tilapia.

This study characterized the local effects of extracellular osmolality and prolactin (PRL) on branchial ionoregulatory function of a euryhaline teleost, Mozambique tilapia (Oreochromis mossambicus). First, gill filaments were dissected from freshwater (FW)-acclimated tilapia and incubated in four different osmolalities, 280, 330, 380, and 450 mosmol/kg H2O. The mRNA expression of Na(+)/K(+)-ATPase α1a (NKA α1a) and Na(+)/Cl(-) cotransporter (NCC) showed higher expression with decreasing media osmolalities, while Na(+)/K(+)/2Cl(-) cotransporter 1a (NKCC1a) and PRL receptor 2 (PRLR2) mRNA levels were upregulated by increases in media osmolality. We then incubated gill filaments in media containing ovine PRL (oPRL) and native tilapia PRLs (tPRL177 and tPRL188). oPRL and the two native tPRLs showed concentration-dependent effects on NCC, NKAα1a, and PRLR1 expression; Na(+)/H(+) exchanger 3 (NHE3) expression was increased by 24 h of incubation with tPRLs. Immunohistochemical observation showed that oPRL and both tPRLs maintained a high density of NCC- and NKA-immunoreactive ionocytes in cultured filaments. Furthermore, we found that tPRL177 and tPRL188 differentially induce expression of these ion transporters, according to incubation time. Together, these results provide evidence that ionocytes of Mozambique tilapia may function as osmoreceptors, as well as directly respond to PRL to modulate branchial ionoregulatory functions.