Development of branchial ionocytes in embryonic and larval stages of cloudy catshark, Scyliorhinus torazame.

In teleost fish, branchial ionocytes are important sites for osmoregulation and acid-base regulation by maintaining ionic balance in the body fluid. During the early developmental stages before the formation of the gills, teleost ionocytes are localized in the yolk-sac membrane and body skin. By comparing with teleost fish, much less is known about ionocytes in developing embryos of elasmobranch fish. The present study investigated the development of ionocytes in the embryo and larva of cloudy catshark, Scyliorhinus torazame. We first observed ionocyte distribution by immunohistochemical staining with anti-Na+/K+-ATPase (NKA) and anti-vacuolar-type H+-ATPase (V-ATPase) antibodies. The NKA- and V-ATPase-rich ionocytes appeared as single cells in the gill filaments from stage 31, the stage of pre-hatching, while the ionocytes on the body skin and yolk-sac membrane were also observed. From stage 32, in addition to single ionocytes on the gill filaments, some outstanding follicular structures of NKA-immunoreactive cells were developed to fill the inter-filament region of the gill septa. The follicular ionocytes possess NKA in the basolateral membrane and Na+/H+ exchanger 3 in the apical membrane, indicating that they are involved in acid-base regulation like single NKA-rich ionocytes. Three-dimensional analysis and whole-mount immunohistochemistry revealed that the distribution of follicular ionocytes was limited to the rostral side of gill septum. The rostral sides of gill septum might be exposed to faster water flow than caudal side because the gills of sharks gently curved backward. This dissymmetric distribution of follicular ionocytes is considered to facilitate efficient body-fluid homeostasis of catshark embryo.

Vitamin D is involved in the regulation of Cl- uptake in zebrafish (Danio rerio).

Cl- is a major anion in the bodily fluids of vertebrates, and maintaining its homeostasis is essential for normal physiological functions. Fishes inhabiting freshwater (FW) passively lose body fluid ions, including Cl-, to the external environment because of the electrochemical gradient of ions across the body surface. Therefore, FW fishes have to actively absorb Cl- from the surroundings to maintain ion homeostasis in their bodily fluids. Hormonal control is vital for modulating ion uptake in fish. Vitamin D is involved in the regulation of Ca2+ uptake and acid secretion in fish. In the present study, we found that the levels of bioactive vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), significantly increased in zebrafish embryos and adults after exposure to water containing low levels of Cl-. Moreover, the administration of 1α,25(OH)2D3 treatment (20 µg/L) in zebrafish embryos, and intraperitoneal (i.p.) injection of 1α,25(OH)2D3 (5 µg/kg body mass) in zebrafish adults, resulting the increased Cl- content in bodily fluid in zebrafish. Na+-Cl- cotransporter 2b (NCC2b) and Cl- channel 2c (CLC2c) are specifically expressed during Cl- uptake by ionocytes in zebrafish. Our results indicated that the mRNA and protein expression of NCC2b and CLC2c considerably increased in the zebrafish with exogenous 1α,25(OH)2D3 treatment. Additionally, exogenous 1α,25(OH)2D3 administration increased the number of NCC2b- and CLC2c-expressing cells in yolk skins of zebrafish embryos and the gill filaments of zebrafish adults. Transcript signals of vitamin D receptors (VDRs) were identified in NCC2b-expressing cells. Knockdown of VDRa and VDRb significantly reduced the expression of NCC2b and CLC2c and the number of NCC2b- and CLC2c-expressing cells. These results indicate that vitamin D can affect Cl- uptake in zebrafish and extend our knowledge of the role of vitamin D in fish physiology.

Impacts of temperature and turbidity on the gill physiology of darter species.

Fish gills are complex organs that have direct contact with the environment and perform numerous functions including gas exchange and ion regulation. Determining if gill morphometry can change under different environmental conditions to maintain and/or improve gas exchange and ion regulation is important for understanding if gill plasticity can improve survival with increasing environmental change. We assessed gill morphology (gas exchange and ion regulation metrics), hematocrit and gill Na+/K+ ATPase activity of wild-captured blackside darter (Percina maculata), greenside darter (Etheostoma blennioides), and johnny darter (Etheostoma nigrum) at two temperatures (10 and 25 °C) and turbidity levels (8 and 94 NTU). Samples were collected August and October 2020 in the Grand River to assess temperature differences, and August 2020 in the Thames River to assess turbidity differences. Significant effects of temperature and/or turbidity only impacted ionocyte number, lamellae width, and hematocrit. An increase in temperature decreased ionocyte number while an increase in turbidity increased lamellae width. Hematocrit had a species-specific response for both temperature and turbidity. Findings suggest that the three darter species have limited plasticity in gill morphology, with no observed compensatory changes in hematocrit or Na+/K+ ATPase activity to maintain homeostasis under the different environmental conditions.

Impact of turbidity on the gill morphology and hypoxia tolerance of eastern sand darter (Ammocrypta pellucida).

Anthropogenic stressors such as agriculture and urbanization can increase river turbidity, which can negatively impact fish gill morphology and growth due to reduced oxygen in the benthic environment. We assessed the gill morphology, field metabolic rate (FMR), and two hypoxia tolerance metrics (oxygen partial pressure at loss of equilibrium, PO2 at LOE, and critical oxygen tension, Pcrit) of eastern sand darter (Ammocrypta pellucida), a small benthic fish listed as threatened under the Species at Risk Act in Canada, from rivers in southern Ontario. Field trials were conducted streamside in the Grand River (August 2019; mean NTU 8) and in the comparatively more turbid Thames River (August 2020; mean NTU 94) to test the effect of turbidity on each physiological endpoint. Gills were collected from incidental mortalities and museum specimens, and were assessed using hematoxylin and eosin and immunofluorescent staining. The between-river comparison indicated that turbidity significantly increased interlamellar space and filament width but had no significant influence on other gill morphometrics or FMR. Turbidity significantly increased PO2 at LOE (i.e., fish had a lower hypoxia tolerance) but did not significantly impact Pcrit. Therefore, although turbidity influences hypoxia tolerance through LOE, turbidity levels were not sufficiently high in the study rivers to contribute to measurable changes in gill morphology or metabolism in the wild. Determining whether changes in gill morphology or metabolism occur under higherturbidity levels would help resolve the ecological importance of turbidity on species physiology in urban and agricultural ecosystems.

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.

Regulation of cell quiescence-proliferation balance by Ca2+-CaMKK-Akt signaling.

Compared with our extensive understanding of the cell cycle, we have limited knowledge of how the cell quiescence-proliferation decision is regulated. Using a zebrafish epithelial model, we report a novel signaling mechanism governing the cell quiescence-proliferation decision. Zebrafish Ca2+-transporting epithelial cells, or ionocytes, maintain high cytoplasmic Ca2+ concentration ([Ca2+]c) due to the expression of Trpv6. Genetic deletion or pharmacological inhibition of Trpv6, or reduction of external Ca2+ concentration, lowered the [Ca2+]c and reactivated these cells. The ionocyte reactivation was attenuated by chelating intracellular Ca2+ and inhibiting calmodulin (CaM), suggesting involvement of a Ca2+ and CaM-dependent mechanism. Long-term imaging studies showed that after an initial decrease, [Ca2+]c gradually returned to the basal levels. There was a concomitant decease in endoplasmic reticulum (ER) Ca2+ levels. Lowering the ER Ca2+ store content or inhibiting ryanodine receptors impaired ionocyte reactivation. Further analyses suggest that CaM-dependent protein kinase kinase (CaMKK) is a key molecular link between Ca2+ and Akt signaling. Genetic deletion or inhibition of CaMKK abolished cell reactivation, which could be rescued by expression of a constitutively active Akt. These results suggest that the quiescence-proliferation decision in zebrafish ionocytes is regulated by Trpv6-mediated Ca2+ and CaMKK-Akt signaling.

Evolving views of ionic, osmotic and acid-base regulation in aquatic animals.

The regulation of ionic, osmotic and acid-base (IOAB) conditions in biological fluids is among the most fundamental functions in all organisms; being surrounded by water uniquely shapes the IOAB regulatory strategies of water-breathing animals. Throughout its centennial history, Journal of Experimental Biology has established itself as a premier venue for publication of comparative, environmental and evolutionary studies on IOAB regulation. This Review provides a synopsis of IOAB regulation in aquatic animals, some of the most significant research milestones in the field, and evolving views about the underlying cellular mechanisms and their evolutionary implications. It also identifies promising areas for future research and proposes ideas for enhancing the impact of aquatic IOAB research.

Subcellular localization of Na+/K+-ATPase isoforms resolved by in situ hybridization chain reaction in the gill of chum salmon at freshwater and seawater.

The Na+/K+-ATPase (NKA) α1-isoforms were examined by in situ hybridization chain reaction (ISHCR) using short hairpin DNAs, and we showed triple staining of NKA α1a, α1b, and α1c transcripts in the gill of chum salmon acclimated to freshwater (FW) and seawater (SW). The NKA α1-isoforms have closely resembled nucleotide sequences, which could not be differentiated by conventional in situ hybridization. The ISHCR uses a split probe strategy to allow specific hybridization using regular oligo DNA, resulting in high specificity at low cost. The results showed that NKA α1c was expressed ubiquitously in gill tissue and no salinity effects were observed. FW lamellar ionocytes (type-I ionocytes) expressed cytoplasmic NKA α1a and nuclear NKA α1b transcripts. However, both transcripts of NKA α1a and α1b were present in the cytoplasm of immature type-I ionocytes. The developing type-I ionocytes increased the cytoplasmic volume and migrated to the distal region of the lamellae. SW filament ionocytes (type-II ionocytes) expressed cytoplasmic NKA α1b transcripts as the major isoform. Results from morphometric analysis and nonmetric multidimensional scaling indicated that a large portion of FW ionocytes was NKA α1b-rich, suggesting that isoform identity alone cannot mark the ionocyte types. Both immature or residual type-II ionocytes and type-I ionocytes were found on the FW and SW gills, suggesting that the chum salmon retains the potential to switch the ionocyte population to fit the ion-transporting demands, which contributes to their salinity tolerance and osmoregulatory plasticity.

Molecular targets of prolactin in mummichogs (Fundulus heteroclitus): Ion transporters/channels, aquaporins, and claudins.

Prolactin (Prl) was identified over 60 years ago in mummichogs (Fundulus heteroclitus) as a "freshwater (FW)-adapting hormone", yet the cellular and molecular targets of Prl in this model teleost have remained unknown. Here, we conducted a phylogenetic analysis of two mummichog Prl receptors (Prlrs), designated Prlra and Prlrb, prior to describing the tissue- and salinity-dependent expression of their associated mRNAs. We then administered ovine Prl (oPrl) to mummichogs held in brackish water and characterized the expression of genes associated with FW- and seawater (SW)-type ionocytes. Within FW-type ionocytes, oPrl stimulated the expression of Na+/Cl- cotransporter 2 (ncc2) and aquaporin 3 (aqp3). Alternatively, branchial Na+/H+ exchanger 2 and -3 (nhe2 and -3) expression did not respond to oPrl. Gene transcripts associated with SW-type ionocytes, including Na+/K+/2Cl- cotransporter 1 (nkcc1), cystic fibrosis transmembrane regulator 1 (cftr1), and claudin 10f (cldn10f) were reduced by oPrl. Isolated gill filaments incubated with oPrl in vitro exhibited elevated ncc2 and prlra expression. Given the role of Aqps in supporting gastrointestinal fluid absorption, we assessed whether several intestinal aqp transcripts were responsive to oPrl and found that aqp1a and -8 levels were reduced by oPrl. Our collective data indicate that Prl promotes FW-acclimation in mummichogs by orchestrating the expression of solute transporters/channels, water channels, and tight-junction proteins across multiple osmoregulatory organs.

Cutaneous respiration and osmoregulation in amphibious fishes.

In his early career, August Krogh made fundamental discoveries of the properties of cutaneous respiration in fish, frogs and other vertebrates. Following Krogh's example, the study of amphibious fishes provides an excellent model to understand how the skin morphology and physiological mechanisms evolved to meet the dual challenges of aquatic and terrestrial environments. The skin of air-exposed fishes takes on many of the functions that are typically associated with the gills of fish in water: gas exchange, gas sensing, iono- and osmoregulation, and nitrogen excretion. The skin of amphibious fishes has capillaries close to the surface in the epidermis. Skin ionocytes or mitochondrial-rich cells (MRCs) in the epidermis are thought to be responsible for ion exchange, as well as ammonia excretion in the amphibious mangrove rivulus Kryptolebias marmoratus. Ammonia gas (NH3) moves down the partial pressure gradient from skin capillaries to the surface through ammonia transporters (e.g., Rhcg) and NH3 is volatilized from the mucus film on the skin. Future studies are needed on the skin of amphibious fishes from diverse habitats to understand more broadly the role of the skin as a multifunctional organ.

Transient receptor potential vanilloid 4 modulates ion balance through the isotocin pathway in zebrafish (Danio rerio).

Isotocin controls ion regulation through modulating the functions of ionocytes (also called mitochondria-rich cells or chloride cells). However, little is known about the upstream molecule of the isotocin system. Herein, we identify transient receptor potential vanilloid 4 (TRPV4), which regulates the mRNA and protein expressions of isotocin and affects ion regulation through the isotocin pathway. Double immunohistochemical results showed that TRPV4 is expressed in isotocinergic neurons in the hypothalamus of the adult zebrafish brain. To further elucidate the roles of TRPV4, we manipulated TRPV4 protein expression and evaluated its ionoregulatory functions in zebrafish embryos. TRPV4 gene knockdown with morpholino oligonucleotides decreased ionic contents (Na+, Cl-, and Ca2+) of whole larvae and the H+-secreting function of larval skin of zebrafish. mRNA expressions of ionocyte-related transporters, including H+-ATPase, the epithelial Ca2+ channel, and the Na+-Cl- cotransporter, were also suppressed in trpv4 morphants. Numbers of ionocytes (H+-ATPase-rich cells and Na+-K+-ATPase-rich cells) and epidermal stem cells in zebrafish larval skin also decreased after trpv4 knockdown. Our results showed that TRPV4 modulates ion balance through the isotocin pathway.

Cortisol is an osmoregulatory and glucose-regulating hormone in Atlantic sturgeon, a basal ray-finned fish.

Our current understanding of the hormonal control of ion regulation in aquatic vertebrates comes primarily from studies on teleost fishes, with relatively little information on more basal fishes. We investigated the role of cortisol in regulating seawater tolerance and its underlying mechanisms in an anadromous chondrostean, the Atlantic sturgeon (Acipenser oxyrinchus). Exposure of freshwater-reared Atlantic sturgeon to seawater (25 ppt) resulted in transient (1-3 day) increases in plasma chloride, cortisol and glucose levels and long-term (6-14 day) increases in the abundance of gill Na+/K+/2Cl- cotransporter (NKCC), which plays a critical role in salt secretion in teleosts. The abundance of gill V-type H+-ATPase, which is thought to play a role in ion uptake in fishes, decreased after exposure to seawater. Gill Na+/K+-ATPase activity did not increase in 25 ppt seawater, but did increase in fish gradually acclimated to 30 ppt. Treatment of Atlantic sturgeon in freshwater with exogenous cortisol resulted in dose-dependent increases in cortisol, glucose and gill NKCC and H+-ATPase abundance. Our results indicate that cortisol has an important role in regulating mechanisms for ion secretion and uptake in sturgeon and provide support for the hypothesis that control of osmoregulation and glucose by corticosteroids is a basal trait of jawed vertebrates.

Respirometry and cutaneous oxygen flux measurements reveal a negligible aerobic cost of ion regulation in larval zebrafish (Danio rerio).

Fishes living in fresh water counter the passive loss of salts by actively absorbing ions through specialized cells termed ionocytes. Ionocytes contain ATP-dependent transporters and are enriched with mitochondria; therefore ionic regulation is an energy-consuming process. The purpose of this study was to assess the aerobic costs of ion transport in larval zebrafish (Danio rerio). We hypothesized that changes in rates of Na+ uptake evoked by acidic or low Na+ rearing conditions would result in corresponding changes in whole-body oxygen consumption (MO2 ) and/or cutaneous oxygen flux (JO2 ), measured at the ionocyte-expressing yolk sac epithelium using the scanning micro-optrode technique (SMOT). Larvae at 4 days post-fertilization (dpf) that were reared under low pH (pH 4) conditions exhibited a higher rate of Na+ uptake compared with fish reared under control conditions (pH 7.6), yet they displayed a lower MO2  and no difference in cutaneous JO2 Despite a higher Na+ uptake capacity in larvae reared under low Na+ conditions, there were no differences in MO2  and JO2  at 4 dpf. Furthermore, although Na+ uptake was nearly abolished in 2 dpf larvae lacking ionocytes after morpholino knockdown of the ionocyte proliferation regulating transcription factor foxi3a, MO2 and JO2  were unaffected. Finally, laser ablation of ionocytes did not affect cutaneous JO2 Thus, we conclude that the aerobic costs of ion uptake by ionocytes in larval zebrafish, at least in the case of Na+, are below detection using whole-body respirometry or cutaneous SMOT scans, providing evidence that ion regulation in zebrafish larvae incurs a low aerobic cost.

Reassessing the contribution of the Na+/H+ exchanger Nhe3b to Na+ uptake in zebrafish (Danio rerio) using CRISPR/Cas9 gene editing.

Freshwater fishes absorb Na+ from their dilute environment using ion-transporting cells. In larval zebrafish (Danio rerio), Na+ uptake is coordinated by (1) Na+/H+ exchanger 3b (Nhe3b) and (2) H+-ATPase-powered electrogenic uptake in H+-ATPase-rich (HR) cells and by (3) Na+-Cl--cotransporter (Ncc) expressed in NCC cells. The present study aimed to better understand the roles of these three proteins in Na+ uptake by larval zebrafish under 'normal' (800 µmol l-1) and 'low' (10 µmol l-1) Na+ conditions. We hypothesized that Na+ uptake would be reduced by CRISPR/Cas9 knockout (KO) of slc9a3.2 (encoding Nhe3b), particularly in low Na+ where Nhe3b is believed to play a dominant role. Contrary to this hypothesis, Na+ uptake was sustained in nhe3b KO larvae under both Na+ conditions, which led to the exploration of whether compensatory regulation of H+-ATPase or Ncc was responsible for maintaining Na+ uptake in nhe3b KO larvae. mRNA expression of the genes encoding H+-ATPase and Ncc was not altered in nhe3b KO larvae. Moreover, morpholino knockdown of H+-ATPase, which significantly reduced H+ flux by HR cells, did not reduce Na+ uptake in nhe3b KO larvae, nor did rearing larvae in chloride-free conditions, thereby eliminating any driving force for Na+-Cl--cotransport via Ncc. Finally, simultaneously treating nhe3b KO larvae with H+-ATPase morpholino and chloride-free conditions did not reduce Na+ uptake under normal or low Na+ These findings highlight the flexibility of the Na+ uptake system and demonstrate that Nhe3b is expendable to Na+ uptake in zebrafish and that our understanding of Na+ uptake mechanisms in this species is incomplete.

The Rhesus glycoprotein Rhcgb is expendable for ammonia excretion and Na+ uptake in zebrafish (Danio rerio).

In zebrafish (Danio rerio), the ammonia-transporting Rhesus glycoprotein Rhcgb is implicated in mechanisms of ammonia excretion and Na+ uptake. In particular, Rhcgb is thought to play an important role in maintaining ammonia excretion in response to alkaline conditions and high external ammonia (HEA) exposure, in addition to facilitating Na+ uptake via a functional metabolon with the Na+/H+-exchanger Nhe3b, specifically under low Na+ conditions. In the present study, we hypothesized that CRISPR/Cas9 knockout of rhcgb would reduce ammonia excretion and Na+ uptake capacity, particularly under the conditions listed above that have elicited increases in Rhcgb-mediated ammonia excretion and/or Na+ uptake. Contrary to this hypothesis, however, larval and juvenile rhcgb knockout (KO) mutants showed no reductions in ammonia excretion or Na+ uptake under any of the conditions tested in our study. In fact, under control conditions, rhcgb KO mutants generally displayed an increase in ammonia excretion, potentially due to increased transcript abundance of another rh gene, rhbg. Under alkaline conditions, rhcgb KO mutants were also able to maintain ammonia excretion, similar to wild-type fish, and stimulation of ammonia excretion after HEA exposure also was not affected by rhcgb KO. Surprisingly, ammonia excretion and Na+ uptake were unaffected by rhcgb or nhe3b KO in juvenile zebrafish acclimated to normal (800 µmol/L) or low (10 µmol/L) Na+ conditions. These results demonstrate that Rhcgb is expendable for ammonia excretion and Na+ uptake in zebrafish, highlighting the plasticity and flexibility of these physiological systems in this species.

Endogenous Notch Signaling in Adult Kidneys Maintains Segment-Specific Epithelial Cell Types of the Distal Tubules and Collecting Ducts to Ensure Water Homeostasis.

BACKGROUND: Notch signaling is required during kidney development for nephron formation and principal cell fate selection within the collecting ducts. Whether Notch signaling is required in the adult kidney to maintain epithelial diversity, or whether its loss can trigger principal cell transdifferentiation (which could explain acquired diabetes insipidus in patients receiving lithium) is unclear. METHODS: To investigate whether loss of Notch signaling can trigger principal cells to lose their identity, we genetically inactivated Notch1 and Notch2, inactivated the Notch signaling target Hes1, or induced expression of a Notch signaling inhibitor in all of the nephron segments and collecting ducts in mice after kidney development. We examined renal function and cell type composition of control littermates and mice with conditional Notch signaling inactivation in adult renal epithelia. In addition, we traced the fate of genetically labeled adult kidney collecting duct principal cells after Hes1 inactivation or lithium treatment. RESULTS: Notch signaling was required for maintenance of Aqp2-expressing cells in distal nephron and collecting duct segments in adult kidneys. Fate tracing revealed mature principal cells in the inner stripe of the outer medulla converted to intercalated cells after genetic inactivation of Hes1 and, to a lesser extent, lithium treatment. Hes1 ensured repression of Foxi1 to prevent the intercalated cell program from turning on in mature Aqp2+ cell types. CONCLUSIONS: Notch signaling viaHes1 regulates maintenance of mature renal epithelial cell states. Loss of Notch signaling or use of lithium can trigger transdifferentiation of mature principal cells to intercalated cells in adult kidneys.

Arginine Vasopressin Modulates Ion and Acid/Base Balance by Regulating Cell Numbers of Sodium Chloride Cotransporter and H+-ATPase Rich Ionocytes.

Arginine vasopressin (Avp) is a conserved pleiotropic hormone that is known to regulate both water reabsorption and ion balance; however, many of the mechanisms underlying its effects remain unclear. Here, we used zebrafish embryos to investigate how Avp modulates ion and acid-base homeostasis. After incubating embryos in double-deionized water for 24 h, avp mRNA expression levels were significantly upregulated. Knockdown of Avp protein expression by an antisense morpholino oligonucleotide (MO) reduced the expression of ionocyte-related genes and downregulated whole-body Cl- content and H+ secretion, while Na+ and Ca2+ levels were not affected. Incubation of Avp antagonist SR49059 also downregulated the mRNA expression of sodium chloride cotransporter 2b (ncc2b), which is a transporter responsible for Cl- uptake. Correspondingly, avp morphants showed lower NCC and H+-ATPase rich (HR) cell numbers, but Na+/K+-ATPase rich (NaR) cell numbers remained unchanged. avp MO also downregulated the numbers of foxi3a- and p63-expressing cells. Finally, the mRNA expression levels of calcitonin gene-related peptide (cgrp) and its receptor, calcitonin receptor-like 1 (crlr1), were downregulated in avp morphants, suggesting that Avp might affect Cgrp and Crlr1 for modulating Cl- balance. Together, our results reveal a molecular/cellular pathway through which Avp regulates ion and acid-base balance, providing new insights into its function.

Na+ K+ ATPase isoform switching in zebrafish during transition to dilute freshwater habitats.

Na+ K+ ATPase (NKA) is crucial to branchial ion transport as it uses the energy from ATP to move Na+ against its electrochemical gradient. When fish encounter extremely dilute environments the energy available from ATP hydrolysis may not be sufficient to overcome thermodynamic constraints on ion transport. Yet many fish species-including zebrafish-are capable of surviving in dilute environments. Despite much study, the physiological mechanisms by which this occurs remain poorly understood. Here, we demonstrate that zebrafish acclimated to less than 10 µM Na+ water exhibit upregulation of a specific NKA α subunit ( zatp1a1a.5) that, unlike most NKA heterotrimers, would result in transfer of only a single Na+ and K+ per ATP hydrolysis reaction. Thermodynamic models demonstrate that this change is sufficient to reduce the activation energy of NKA, allowing it to overcome the adverse electrochemical gradient imposed by dilute freshwater. Importantly, upregulation of zatp1a1a.5 also coincides with the recovery of whole body Na+ post-transfer, which occurs within 24 h. While these structural modifications are crucial for allowing zebrafish to survive in ion-poor environments, phylogenetic and structural analysis of available α subunits from a range of teleosts suggests this adaptation is not widely distributed.

Heterogeneity of Pulmonary Stem Cells.

Epithelial stem cells reside within multiple regions of the lung where they renew various region-specific cells. In addition, there are multiple routes of regeneration after injury through built-in heterogeneity within stem cell populations and through a capacity for cellular plasticity among differentiated cells. These processes are important facets of respiratory tissue resiliency and organism survival. However, this regenerative capacity is not limitless, and repetitive or chronic injuries, environmental stresses, or underlying factors of disease may ultimately lead to or contribute to tissue remodeling and end-stage lung disease. This chapter will review stem cell heterogeneity among pulmonary epithelia in the lower respiratory system, discuss recent findings that may challenge long-held scientific paradigms, and identify several clinically relevant research opportunities for regenerative medicine.

Prolactin controls branchial clcn2c but not atp1a1a.2 in zebrafish Danio rerio.

This study examined the branchial epithelium of stenohaline zebrafish Danio rerio, and in particular Na+ -Cl- cotransporter-like 2 (Slc12a10.2)-expressing ionocytes (Na+ -Cl- cotransporter [Ncc]-cells), which mediate the active uptake of ions from freshwater environments. The study assessed whether the pituitary hormone prolactin (Prl) stimulates the expression of messenger (m)RNAs encoding a Clc Cl- channel family member (clcn2c) and a Na+ -K+ -ATPase α1 subunit (atp1a1a.2) expressed in Ncc-cells. Branchial clcn2c, but not atp1a1a.2 levels, were sensitive to Prl both in vitro and in vivo. These observations suggest that Prl contributes to maintaining systemic Cl- balance via the regulation of clcn2c.