- Email: [email protected]
Neuroendocrine control of ionic balance in zebrafish
Accepted Manuscript Neuroendocrine control of ionic balance in zebrafish Raymond W.M. Kwong, Yusuke Kumai, Steve F. Perry PII: DOI: Reference:
S0016-6480(16)30137-X http://dx.doi.org/10.1016/j.ygcen.2016.05.016 YGCEN 12404
To appear in:
General and Comparative Endocrinology
Received Date: Revised Date: Accepted Date:
4 October 2015 6 May 2016 11 May 2016
Please cite this article as: Kwong, R.W.M., Kumai, Y., Perry, S.F., Neuroendocrine control of ionic balance in zebrafish, General and Comparative Endocrinology (2016), doi: http://dx.doi.org/10.1016/j.ygcen.2016.05.016
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1
Neuroendocrine control of ionic balance in zebrafish Raymond W.M. Kwong*, Yusuke Kumai1, Steve F. Perry
Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5
*Corresponding author: Raymond W.M. Kwong, Ph.D. Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5 Tel. 1-613-562-5800 ext 6010 E-mail: [email protected]
1
Dr. Kumai passed away suddenly in August 2015
2 Abstract Zebrafish (Danio rerio) is an emerging model for integrative physiological research. In this minireview, we discuss recent advances in the neuroendocrine control of ionic balance in this species, and identify current knowledge gaps and issues that would benefit from further investigation. Zebrafish inhabit a hypo-ionic environment and therefore are challenged by a continual loss of ions to the water. To maintain ionic homeostasis, they must actively take up ions from the water and reduce passive ion loss. The adult gill or the skin of larvae are the primary sites of ionic regulation. Current models for the uptake of major ions in zebrafish incorporate at least three types of ion transporting cells (also called ionocytes); H+-ATPase-rich cells for Na+ uptake, Na+/K+-ATPase-rich cells for Ca2+ uptake, and Na+/Cl--cotransporter expressing cells for both Na+ and Cl- uptake. The precise molecular mechanisms regulating the paracellular loss of ions remain largely unknown. However, epithelial tight junction proteins, including claudins, are thought to play a critical role in reducing ion losses to the surrounding water. Using the zebrafish model, several key neuroendocrine factors were identified as regulators of epithelial ion movement, including the catecholamines (adrenaline and noradrenaline), cortisol, the reninangiotensin system, parathyroid hormone and prolactin. Increasing evidence also suggests that gasotransmitters, such as H2S, are involved in regulating ion uptake.
Keywords: Calcium; cortisol; ionic regulation; ionocyte; neuroendocrine control; sodium
3 1. Introduction Previous studies using traditional models [e.g. rainbow trout (Oncorhynchus mykiss), goldfish (Carassius auratus), killifish (Fundulus heteroclitus) and American eel (Anguilla rostrata)] have provided important information on the fundamental mechanisms of osmoregulation in fish. However, limitations in the use of molecular physiological approaches in these species have hindered precise characterization of the underlying molecular pathways and mechanisms. The zebrafish (Danio rerio) has emerged as an important model for integrative physiological research, owing to the availability of genetic databases, applicability of genetic editing and manipulation, and transparency of the embryos allowing direct cellular/tissue observations. The ion transport pathways in zebrafish are thought to resemble those occurring in different segments of the mammalian kidney (Hwang and Chou, 2013). For example, the expression of different transporters for the absorption of Na+, Ca2+ and Cl-, and their regulatory functions, appear to be conserved between zebrafish gills/skin and mammalian kidney. Thus, the zebrafish is a useful model system for advancing our understanding of the ion-regulatory functions in the mammalian kidney. Several previous studies using zebrafish have identified key neuroendocrine factors which are important in regulating ionic homeostasis and transepithelial ion movements. The regulation of ion movement in zebrafish was described in several previous reviews (Hwang, 2009; Hwang and Perry, 2010; Hwang and Chou, 2013; Kwong et al., 2014a). The present review provides a brief overview of the mechanisms of ionic regulation in zebrafish and discusses some of the recent advances surrounding their neuroendocrine control. More specifically, we focus on the adrenergic and glucocorticoid systems, angiotensin II, parathyroid hormone (PTH), prolactin and gasotransmitters. Finally, this review reveals current knowledge gaps and identifies issues that could be addressed in future studies.
4 2. Molecular physiology of ionic regulation in zebrafish Zebrafish, a freshwater teleost, are hyper-ionic to their environment and thus are challenged constantly by diffusive ion losses. To maintain whole body ionic homeostasis, they need to actively absorb ions from, and reduce passive ion losses to, the environment. In adults, the gills are the predominant site for active ion uptake through specific ion-transporting cells termed ionocytes. During larval stages before the gills are fully developed, regulation of epithelial ion transport is primarily mediated by the ionocytes found on the skin of the yolk sac. Although ionocytes are recognized as the universal site of ionic uptake in fish, there is no common nomenclature that yet has been developed to designate their structure and function. Indeed, the development of a common nomenclature is challenging because of interspecific differences in ionocyte subtype that are known to exist across species (for a review, see Dymowska et al., 2012). Current models for the absorption of the major ions (i.e. Na+, Ca2+ and Cl-) in zebrafish incorporate at least 3 types of ionocytes; H+-ATPase-rich cells (HRCs), Na+/K+-ATPase-rich cells (NaRCs) and Na+/Cl--cotransporter-expressing cells (NCCCs) (Figure 1). HRCs express H+-ATPase and Na+/H+-exchanger at the apical membrane (i.e. NHE3b) for H+ secretion and Na+ uptake, respectively; NCCCs express Na+/Cl--cotransporter for both Na+ and Cl- uptake; a subset of NaRCs express epithelial Ca2+ channel (ECaC) at the apical membrane for Ca2+ uptake, and Na+/Ca2+-exchanger and Ca2+-ATPase at the basolateral membrane for Ca2+ extrusion. Different paralogues of Na+/K+-ATPase are expressed at the basolateral membrane in all these 3 types of ionocytes (Horng et al., 2007; Kumai and Perry, 2011; Liao et al., 2009; Pan et al., 2005; Shih et al., 2012; Wang et al., 2009). Recently, the acid-sensing ion channels (ASICs), the closest relatives of the mammalian epithelial Na+ channels (ENaC), were identified at the apical membrane of HRCs in adult zebrafish gills (Dymowska et al., 2015).
5 Paracellular movement of ions and fluid is regulated predominantly by the tight junction proteins. Occludin, claudins, tricellulins and junctional adhesion molecule are major components of the tight junctions. However, claudins are thought to be the primary determinant of the epithelial barrier functions in vertebrates (Turksen and Troy, 2004). To date, there are over 50 different isoforms of claudins that have been identified in zebrafish, and most of them are expressed in a cell- or tissue-specific manner (Baltzegar et al., 2013; Clelland and Kelly, 2010; Kumai et al., 2011; Kwong and Perry, 2013b). The functional characteristics of most of the claudins remain largely unknown. Nonetheless, certain claudin isoforms appear to form selective pores or barriers to regulate the paracellular movement of ions or fluid (Bagnat et al., 2007; Kwong et al., 2013; Kwong and Perry, 2013b; Zhang et al., 2010), and are critical for development and osmoregulation (for a review, see Kolosov et al., 2013). 3. Neuroendocrine control of ionic regulation in zebrafish 3.1 Catecholamines In vertebrates, the catecholamines, adrenaline and noradrenaline, are essential for regulating cardiovascular and respiratory functions. It is also documented that the adrenergic system is involved in osmoregulation and acid-base balance in fish (Donald, 1989; Kumai et al., 2012b; Marshall et al., 1993; McDonald et al., 1989; Perry et al., 1984; Perry et al., 1996; Perry and Vermette, 1987; Reid et al., 1998; Vermette and Perry, 1987). Catecholamines either can interact with target cells after their secretion as neurotransmitters from nearby nerve endings, or after their release from chromaffin cells for more widespread distribution via the circulatory system. Using high-performance liquid chromatography (HPLC), catecholamines were detectable in whole embryos at 1 h post fertilization (hpf), presumably derived from maternal transfer (Steele et al., 2011). At 2 days post fertilization (dpf), chromaffin cells were found to be
6 dispersed as clusters in the inter-renal organ, which then converged to midline by 3 dpf; the chromaffin cells stay in intimate contact with steroidogenic cells throughout the subsequent development (Liu, 2007; To et al., 2007). In zebrafish, both HRCs and NaRCs are innervated (Jonz and Nurse, 2006; Kumai et al., 2012b). Using fluorescently labeled propranolol (nonselective β-adrenergic receptor antagonist), β receptors were found to be distributed in HRCs, as well as in mitochondrion-rich ionocytes (likely NaRCs). The ecac-expressing NaRCs also are closely associated with an extensive network of nerves (Figure 2). These observations suggest the possible direct control of ionocyte functions by the neuronal systems in zebrafish. Using pharmacological approaches, it was demonstrated that blockade of β-adrenergic receptors decreased Na+ uptake in larval zebrafish, whereas blockade of α-adrenergic receptors increased Na+ uptake (Kumai et al., 2012b). Similarly, pharmacological inhibition of β- and αadrenergic receptors decreased and increased Ca2+ uptake, respectively (R.W.M Kwong and S.F. Perry; unpublished results). Therefore, it appears that β- and α-adrenergic systems play opposing roles in regulating Na+ and Ca2+ uptake. More importantly, knockdown of specific β-adrenergic receptors was shown to inhibit Na+ uptake in larval zebrafish exposed to acidic or ion-poor water, conditions known to increase Na+ transport capacity in this species (Kumai et al., 2012b). These observations suggest that the increased capacity for Na+ uptake is, at least in part, mediated by activation of the β-adrenergic system. Interestingly, Na+ uptake appears to be regulated by different β receptors under different environmental conditions (i.e. β1 or β2b receptors are involved in increasing Na+ uptake during exposure to acidic water, while β2a receptors play a role in ion-poor conditions) (Kumai et al., 2012b). However, the precise molecular pathways underlying the increased Na+ uptake and the intracellular signalling cascade are yet to be fully
7 characterized. The specific role of different subtypes of β- or α-receptors in modulating ion uptake also requires further investigation. 3.2 Cortisol Cortisol is involved in regulating a diverse range of biological processes in fish, including reproduction, metabolism, immune responses and osmoregulation (Mommsen et al., 1999). Similar to some other freshwater species, exposure of zebrafish to cortisol stimulates the uptake of both Na+ and Ca2+ (Kumai et al., 2012a; Lin et al., 2016; Lin et al., 2011), and the secretion of H+ (Lin et al., 2015). Using in situ hybridization or immunohistochemistry, the glucocorticoid receptor (GR) was found to be expressed in NaRCs (Cruz et al., 2013), HRCs (Kumai et al., 2012a) and NCCCs (Lin et al., 2016) in zebrafish. Exposure to cortisol was also shown to increase mRNA expression of genes that are involved in ion uptake or H+ secretion, including ecac, ncc, nhe3b, atp6v1a, ae1b, ca2-like a, and ca15a (Lin et al., 2015; Lin et al., 2011). Additionally, morpholino knockdown of the GR prevented the increase in mRNA expression of ecac and ncc caused by cortisol treatment (Lin et al., 2016; Lin et al., 2011). Cortisol also modulates the mRNA expression of enzymes/hormone known to regulate Ca2+ uptake, including reducing the hypocalcemic hormone stanniocalcin (stc-1) (Kumai et al., 2014b) and increasing several enzymes responsible for vitamin-D synthesis (Lin et al., 2012). Furthermore, it was demonstrated that cortisol treatment stimulated the mRNA expression of glial cell missing-2 (gcm2) (Kumai et al., 2014b) and forkhead box I3 (foxi3a and foxi3b) (Cruz et al., 2013a), which are transcription factors critically involved in ionocyte development in zebrafish (Chang et al., 2009; Kumai et al., 2014b; Shono et al., 2011). Similarly, GR knockdown was shown to decrease the numbers of HRCs and NaRCs (Cruz et al., 2013b).
8 Together, these studies clearly demonstrate that cortisol plays an important role in promoting ion uptake and ionocyte differentiation in zebrafish. In zebrafish, cortisol appears to be involved in promoting ionic balance during exposure to environmental stressors. For example, the mRNA expression of GR was found to increase in larval zebrafish acclimated to softwater (i.e. low Ca2+ water) (Lin et al., 2011). The mRNA levels of 11β-hydroxylase, a vital enzyme responsible for cortisol synthesis, also was increased in zebrafish acclimated to softwater (Lin et al., 2011). Additionally, whole body cortisol levels in zebrafish were elevated following exposure to low Na+ (Lin et al., 2016) or acidic (pH 4.0) water (Kumai et al., 2012a). In acidic water, the elevated cortisol levels appear to promote Na+ balance by increasing transcellular Na+ uptake as well as by reducing paracellular Na+ losses, probably via activation of the cortisol-GR signalling cascade (Kumai et al., 2012a; Kwong and Perry, 2013a). Cortisol likely stimulates Na+ uptake through the NHE3b–Rhcg1 functional metabolon (Kumai et al., 2012a). The exact mechanisms for the reduction in paracellular Na+ loss by cortisol are not fully understood, but appear to be associated with an increase in the abundance of epithelial tight junction proteins (Kwong and Perry, 2013a). Overall, these findings demonstrate that the cortisol-GR signalling cascade is involved in promoting ionic balance in zebrafish. Interestingly, cortisol is reported to act as a hypocalcemic factor in mammals (Lukert and Raisz, 1990), suggesting different roles for cortisol in ionic regulation in mammals and zebrafish. In other fish species, cortisol is reported to interact with other hormonal systems known to modulate osmoregulation, such as growth hormone and insulin-like growth factor (Mommsen et al., 1999); whether similar interactions also occur in zebrafish remain unclear, and would be an important area for future investigation. 3.3 Angiotensin II
9 In mammals, the renin–angiotensin system (RAS) is involved in regulating salt reabsorption in the kidney (Crowley and Coffman, 2012). Angiotensinogen is first cleaved to angiotensin-I (ANG-I) by the enzyme renin, which is converted to the biologically active form ANG-II by angiotensin-converting enzyme (ACE). The physiological role of the RAS in fish is not completely understood. Nevertheless, increasing evidence suggests that the RAS is involved in osmoregulation (Fuentes and Eddy, 1997; Hoshijima and Hirose, 2007; Kumai et al., 2014a; Smith et al., 1991). It is well documented that ANG-II promotes drinking in euryhaline fish (Beasley et al., 1986; Carrick and Balment, 1983; Fuentes and Eddy, 1996; Kobayashi et al., 1983), potentially increasing salt absorption from the gut. In the European eel Anguilla anguilla, receptors for ANG-II are located in gill ionocytes, and treatment of eels with ANG-II caused an increase in the activity of Na+/K+-ATPase (Marsigliante et al., 1997). In larval zebrafish, ANG-II receptor-like 1a (agtrl1a) is expressed in multiple tissues, including developing lens, otic vesicles and venous vasculature (Tucker et al., 2007). It is unknown whether ANG-II receptors are expressed in ionocytes of zebrafish larvae or adults. Previous studies have provided evidence that the RAS promotes ion uptake in zebrafish. For example, acclimation of larval zebrafish to ion-poor water increased the mRNA expression of renin (Hoshijima and Hirose, 2007). Additionally, whole body ANG-II levels were elevated in zebrafish exposed to ion-poor or acidic water (Kumai et al., 2014a). Importantly, translational gene knockdown of renin substantially attenuated the increased Na+ uptake following acute exposure to acidic or ion-poor water (Kumai et al., 2014a), indicating a role for the RAS in promoting Na+ influx. The downstream signalling pathways in the acute regulation of Na+ uptake by the RAS remain unknown. In fish, ANG-II is a potent stimulant for cortisol synthesis (Arnold-Reed and Balment, 1994), and cortisol is known to promote Na+ uptake in larval
10 zebrafish (discussed above). Interestingly, the increase in Na+ uptake associated with acute acid or ion-poor water exposure was not affected by GR knockdown in larval zebrafish, implying cortisol is not involved in RAS-mediated Na+ regulation (Kumai et al., 2014a). The precise Na+transport pathways which are regulated by the RAS also remain unclear. However, treatment with exogenous ANG-II was found to increase the mRNA expression of NCC (Kumai et al., 2014a). Although ANG-II appears to play a role in stimulating Na+ uptake, the identification of ANG-II receptors in ionocytes or in osmoregulatory tissues, and the underlying molecular mechanisms, have yet to be investigated in zebrafish. 3.4 Parathyroid hormone In mammals, PTH is expressed and secreted predominantly by the parathyroid gland. PTH plays an important role in regulating Ca2+ metabolism and skeletal remodeling. Fish do not have parathyroid glands, and the gill has been proposed to be the evolutionary precursor of the mammalian parathyroid glands (Okabe and Graham, 2004). Zebrafish express two PTH paralogs, PTH1 and PTH2 (Hogan et al., 2005). Their mRNA expression have been detected in various tissues in adults (Lin et al., 2014), and primarily in the central nervous system in larvae (Hogan et al., 2005). Three PTH receptors (PTH1R-a, PTH1R-b and PTH2R) have been identified in zebrafish (Hoare et al., 2000; Rubin and Jüppner, 1999), which appear to be expressed in a tissue-specific manner in adults (Kwong and Perry, 2015a). Similar to mammals, PTH functions as a hypercalcemic hormone and regulates skeletogenesis in zebrafish. To date, no PTH receptor has been identified in zebrafish ionocytes. However, acclimation of larval zebrafish to ion-poor or low Ca2+ water was shown to increase the mRNA expression of pth1 (Hoshijima and Hirose, 2007; Kwong and Perry, 2015a; Lin et al.,
11 2014). Such modulation is likely mediated via the extracellular Ca2+-sensing receptor expressed in ionocytes and/or corpuscles of Stannius (Kwong et al., 2014b; Lin et al., 2014). Additionally, translational gene knockdown of PTH1 reduced whole body Ca2+ levels and decreased mRNA expression of ecac in larval zebrafish (Kwong and Perry, 2015a; Lin et al., 2014). PTH1 also appears to be important in promoting ionocyte differentiation and cartilage development, probably through its interaction with the cell-fate transcription factor gcm2 (Kwong and Perry, 2015a). Furthermore, overexpression of the hypocalcemic hormone calcitonin was found to increase the mRNA expression of PTH receptors in zebrafish (Lafont et al., 2011), suggesting a compensatory role of PTH in maintaining Ca2+ balance. In some other fish species, the PTHrelated protein (PTHrP; a protein member of the PTH family) also was suggested to be an essential hypercalcaemic factor (Abbink and Flik, 2007). Similar to PTH, PTHrP is reported to be critical for skeletogenesis in developing zebrafish (Yan et al., 2012). Whether PTHrP also regulates Ca2+ acquisition in zebrafish remains to be determined. 3.5 Prolactin Several previous studies have suggested that prolactin is important in ionic regulation in zebrafish. In larval zebrafish, prolactin is expressed in the anterior pituitary (Liu et al., 2006; Sbrogna et al., 2003), and its receptors (prlra and prlrb) are abundantly expressed in both the developing gills and kidney (Shu et al., 2016). In adult zebrafish, these receptors are expressed in osmoregulatory organs, including the gills, kidney and intestine (Breves et al., 2013b). It was demonstrated that larval zebrafish exposed to dilute freshwater exhibited an increase in the mRNA expression of prolactin (Hoshijima and Hirose, 2007). Intraperitoneal injection of ovine prolactin increased mRNA expression of ncc and prolactin receptor-a (prlra) in adult zebrafish gills (Breves et al., 2013a). Results from whole-mount in situ hybridization also revealed that the
12 number of ncc-positive cells was reduced in larval zebrafish experiencing NCC knockdown (Breves et al., 2014). Using a line of prolactin-knockout mutants, it was further demonstrated that prolactindeficient larvae could not survive to the adult stage when they were raised in typical freshwater (2 mOsm/L) (Shu et al., 2016). The prolactin-deficient larvae at 6 dpf had lower whole body levels of Na+, K+ and Cl− when compared to the wild-type fish (Shu et al., 2016). Interestingly, the reduction in whole body ion levels in the prolactin-deficient zebrafish was prevented when they were raised in ion-enriched water (163 mOsm/L), allowing them to survive into adults (Shu et al., 2016). Additionally, a decrease in the mRNA expression levels of ncc but increased expression levels of atp1a1a.5 (an HRC marker) was observed in prolactin-deficient zebrafish larvae (Shu et al., 2016). Overall, these studies demonstrate an important role of prolactin in maintaining ionic balance and survival during early development, at least in part via its regulation of the expression of NCC. However, whether prolactin regulates NCC expression directly, or differentiation of NCCCs from ionocyte progenitors is unclear. Future studies should address the underlying mechanisms linking prolactin and ionocyte development and the role of prolactin in regulating NCC function. Prolactin is proposed to be a freshwater-adapting hormone not only by promoting ion uptake, but also by reducing water permeability (Sakamoto and McCormick, 2006). The zebrafish model may prove useful in elucidating the potential role of prolactin in regulating epithelial permeability and the associated downstream signalling processes. 3.6 Gasotransmitters
13 Gasotransmitters, including nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S), are endogenously produced gaseous signalling molecules which regulate a variety of physiological functions in vertebrates, including the cardiovascular, respiratory, nervous and immune systems (Mustafa et al., 2009). Increasing evidence suggests that gasotransmitters may play a role in osmoregulation in fish (Ebbesson et al., 2005; Kumai et al., 2014c; Kwong and Perry, 2015b; Tipsmark and Madsen, 2003). To date, only the role of H2S in ionic regulation has been investigated in zebrafish. Biosynthesis of H2S is predominantly mediated by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), and in adult zebrafish, mRNA expression of cbsb and cse are detected in various tissues, including the gill, intestine and kidney (Kwong and Perry, 2015b). Both CBSb and CSE are expressed in HRCs and NaRCs in larval zebrafish (Kumai et al., 2014c; Kwong and Perry, 2015b), suggesting a capacity of these ionocytes to produce H2S. It was demonstrated that exposure to exogenous H2S donors, either Na2S or GYY4137 (a slow-releasing H2S donor; Lee et al., 2011) substantially reduced Na+ uptake in larval zebrafish (Kumai et al., 2014c). Using pharmacological agents or genetic knockdown approaches, it was shown that inhibition of the H2S-synthesising enzymes significantly attenuated the capacity of fish to lower their Na+ uptake after exposure to Na+-enriched water (Kumai et al., 2014c). These findings indicate that H2S is a physiologically relevant signaling molecule which contributes to reducing Na+ uptake capacity in developing zebrafish. The precise molecular targets for H2S have not been fully characterized. However, treatment with H2S donors did not reduce Na+ uptake in fish lacking HRCs (Kumai et al., 2014c), suggesting H2S interacts with HRCs in reducing Na+ uptake. Interestingly, as opposed to its inhibitory role in Na+ uptake, H2S appears to stimulate Ca2+ uptake in larval zebrafish. For example, exposure to H2S donors Na2S or GYY4137
14 increased Ca2+ influx and whole body Ca2+ content in larval zebrafish (Kwong and Perry, 2015b). Additionally, gene knockdown of CBSb significantly reduced Ca2+ influx in larval zebrafish acclimated to a low Ca2+ environment (Kwong and Perry, 2015b). The mRNA expression of cbsb was also found to increase in fish acclimated to a low Ca2+ environment (Kwong and Perry, 2015b). The exact mechanisms for the stimulatory effects of H2S on Ca2+ influx are not clear, but appear to involve post-translational activation of ECaC via the cAMP-PKA pathways (Kwong and Perry, 2015b). The significance of H2S in reducing Na+ influx while simultaneously increasing Ca2+ influx is unknown. However, these observations suggest that H2S regulates Na+ and Ca2+ uptake through different mechanisms in different cell types. To fully address the physiological relevance of H2S, future studies should examine the levels of endogenous H2S and/or activity of H2S-producing enzymes in response to different environmental conditions, and the effects of H2S on different ion channels/transporters in the various ionocyte subtypes. The potential role of other gasotransmitters (e.g. NO and CO) in ionic regulation, the precise molecular targets for gasotransmitters, and the intracellular signalling pathways, are worthwhile areas for future research. 3.7 Other neuroendocrine factors Several other hormones, such as endothelin, stanniocalcin, isotocin and atrial natriuretic peptide (ANP) are suggested to be involved in regulating ion uptake, ionocyte development and/or acidbase balance in zebrafish. For example, endothelin and stanniocalcin were found to play a critical role in stimulating H+ secretion (Guh et al., 2014) and reducing Ca2+ uptake (Tseng et al., 2009), respectively. In adult zebrafish, isotocin mRNA is predominantly expressed in the brain but also is expressed in other organs including the gill, muscle and ovaries (Chou et al., 2011). Receptors for isotocin (both itnpr-like 1 and itnpr-like 2) are ubiquitously expressed in adults tissues (Chou
15 et al., 2011). In rainbow trout Oncorhynchus mykiss (Kulczykowska, 1997) and sea bream Sparus aurata (Kleszczynska et al., 2006), isotocin levels increase with increasing salinity, suggesting its possible role in hyperosmotic acclimation. Interestingly, mRNA levels of isotocin in larval zebrafish were significantly higher following their exposure to deionised water (Chou et al., 2011). Additionally, gene knockdown of isotocin substantially reduced the whole-body levels of Na+, Ca2+ and Cl- (Chou et al., 2011). Isotocin also appears to be important in promoting the development of various ionocytes, probably through its interaction with epidermal stem cells and foxi3a-expressing ionocyte progenitors (Chou et al., 2011). These findings suggest that in zebrafish, isotocin plays a critical role in promoting ion retention and ionocyte development. A substantial body of evidence suggests that ANP is a seawater-adapting hormone in fish. For example, in the Japanese eel Anguilla japonica, plasma levels of ANP increase after transferring from freshwater to seawater, and treatment with ANP inhibited intestinal Na+ uptake (Kaiya and Takei, 1996; Loretz and Takei, 1997). In larval zebrafish, the expression of anp was confined to the atrium (Berdougo et al., 2003). Interestingly, anp expression was found to substantially increase in larvae following exposure to ion-poor water (Hoshijima and Hirose, 2007). Clearly, the functional role of ANP in zebrafish osmoregulation is virtually unknown and warrants further study. 4. Conclusions and perspectives The zebrafish has emerged as a powerful model vertebrate to study the neuroendocrine control of ionic regulation. Several key neuroendocrine factors and their downstream regulatory pathways have been elucidated using the zebrafish model (Figure 3). These findings have provided new insights into the molecular physiology of ionic regulation in fish. However, the response of these
16 neuroendocrine factors to environmental challenges (e.g. ion-poor water), with a few exceptions (cortisol, ANG II), are largely unknown, and should be addressed in future research. Currently, reverse genetics approaches, particularly the morpholino technology, are used routinely in larval zebrafish to investigate ion regulatory functions. However, the morpholino technique only allows transient gene knockdown during early developmental stages, and is not suitable for assessing gene function in adults. Moreover, there are obvious physiological differences in the ionregulatory systems between larval and adult fish (e.g. ion regulation occurs primarily at the gill, intestine and kidney in adults, whereas it occurs in the skin of yolk sac in larvae). Therefore, larvae and adults may employ different strategies to maintain ionic balance. It is important that future studies examine the regulation of ionic balance (as well as other physiological functions) in adult animals. Recent advances on the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems (Hwang et al., 2013) will undoubtedly prove useful in assessing mechanisms of ion regulation in adults. Despite the obvious advantages, there are also some significant limitations associated with using zebrafish as a model for piscine osmoregulation. For example, the relatively small size and lack of euryhalinity in zebrafish hinder certain experimental procedures, such as evaluation of blood chemistry and effects of osmotic stress. Thus, a continuing comparative approach aimed at assessing inter-specific similarities and differences in ion-regulatory mechanisms arguably is the preferred approach to shed light on the diverse strategies employed by fish to achieve ionic and osmotic homeostasis. 5. Acknowledgements
17 Our original research was financially supported by Natural Sciences and Engineering Research Council (NSERC) Discovery and NSERC Research Tools and Instrumentation Grants to S.F. Perry.
18 Reference Abbink, W., Flik, G., 2007. Parathyroid hormone-related protein in teleost fish. Gen. Comp. Endocrinol. 152, 243-251. Arnold-Reed, D.E., Balment, R.J., 1994. Peptide Hormones Influence in Vitro Interrenal Secretion of Cortisol in the Trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 96, 85-91. Bagnat, M., Cheung, I.D., Mostov, K.E., Stainier, D.Y.R., 2007. Genetic control of single lumen formation in the zebrafish gut. Nat. Cell Biol. 9, 954-960. Baltzegar, D.A., Reading, B.J., Brune, E.S., Borski, R.J., 2013. Phylogenetic revision of the claudin gene family. Marine Genomics 11, 17-26. Beasley, D., Shier, D.N., Malvin, R.L., Smith, G., 1986. Angiotensin-stimulated drinking in marine fish. Am. J. Physiol. 250, R1034-1038. Berdougo, E., Coleman, H., Lee, D.H., Stainier, D.Y.R., Yelon, D., 2003. Mutation of weak atrium/atrial myosin heavy chain disrupts atrial function and influences ventricular morphogenesis in zebrafish. Development 130, 6121-6129. Carrick, S., Balment, R.J., 1983. The renin-angiotensin system and drinking in the euryhaline flounder, Platichthys flesus. Gen. Comp. Endocrinol. 51, 423-433. Chang, W.J., Horng, J.L., Yan, J.J., Hsiao, C.D., Hwang, P.P., 2009. The transcription factor, glial cell missing 2, is involved in differentiation and functional regulation of H+-ATPase-rich cells in zebrafish (Danio rerio). Am. J. Physiol. -Reg. I. 296, R1192-R1201.
19 Chou, M.-Y., Hung, J.-C., Wu, L.-C., Hwang, S.-P., Hwang, P.-P., 2011. Isotocin controls ion regulation through regulating ionocyte progenitor differentiation and proliferation. Cell. Mol. Life Sci. 68, 2797-2809. Clelland, E.S., Kelly, S.P., 2010. Tight junction proteins in zebrafish ovarian follicles: Stage specific mRNA abundance and response to 17β-estradiol, human chorionic gonadotropin, and maturation inducing hormone. Gen. Comp. Endocrinol. 168, 388-400. Crowley, S.D., Coffman, T.M., 2012. Recent advances involving the renin–angiotensin system. Exp. Cell Res. 318, 1049-1056. Cruz, S.A., Chao, P.-L., Hwang, P.-P., 2013a. Cortisol promotes differentiation of epidermal ionocytes through Foxi3 transcription factors in zebrafish (Danio rerio). Comp. Biochem. Physiol. -A 164, 249-257. Cruz, S.A., Lin, C.-H., Chao, P.-L., Hwang, P.-P., 2013b. Glucocorticoid receptor, but not mineralocorticoid Receptor, mediates cortisol regulation of epidermal Ionocyte development and Ion transport in zebrafish (Danio Rerio). PLoS ONE 8, e77997. Donald, J.A., 1989. Adrenaline and branchial nerve stimulation inhibit 45Ca influx into the gills of rainbow trout, Salmo Gairdneri. Journal of Experimental Biology 141, 441-445. Dymowska, A.K., Boyle, D., Schultz, A.G., Goss, G.G., 2015. The role of acid-sensing ion channels (ASICs) in epithelial Na+ uptake in adult zebrafish (Danio rerio). J. Exp. Biol. Dymowska, A.K., Hwang, P.-P., Goss, G.G., 2012. Structure and function of ionocytes in the freshwater fish gill. Res. Physiol. Neurobiol. 184, 282-292.
20 Ebbesson, L.O., Tipsmark, C.K., Holmqvist, B., Nilsen, T., Andersson, E., Stefansson, S.O., Madsen, S.S., 2005. Nitric oxide synthase in the gill of Atlantic salmon: colocalization with and inhibition of Na+,K+-ATPase. J. Exp. Biol. 208, 1011-1017. Fuentes, J., Eddy, F.B., 1996. Drinking in Freshwater-Adapted Rainbow Trout Fry, Oncorhynchus mykiss (Walbaum), in Response to Angiotensin I, Angiotensin II, AngiotensinConverting Enzyme Inhibition, and Receptor Blockade. Physiological Zoology 69, 1555-1569. Fuentes, J., Eddy, F.B., 1997. Drinking in marine, euryhaline and freshwater teleost fish, in: N. Hazon, F.B. Eddy, G. Flik (Eds.), Ionic Regulation in Animals: A Tribute to Professor W.T.W.Potts. Springer Berlin Heidelberg, 135-149. Guh, Y.J., Tseng, Y.C., Yang, C.Y., Hwang, P.P., 2014. Endothelin-1 regulates H+-ATPasedependent transepithelial H+ secretion in zebrafish. Endocrinology 155, 1728-1737. Hoare, S.R.J., Rubin, D.A., Jüppner, H., Usdin, T.B., 2000. Evaluating the ligand specificity of zebrafish parathyroid hormone (PTH) receptors: Comparison of PTH, PTH-Related protein, and tuberoinfundibular peptide of 39 residues. Endocrinology 141, 3080-3086. Hogan, B.M., Danks, J.A., Layton, J.E., Hall, N.E., Heath, J.K., Lieschke, G.J., 2005. Duplicate zebrafish pth genes are expressed along the lateral line and in the central nervous system during embryogenesis. Endocrinology 146, 547-551. Horng, J.-L., Lin, L.-Y., Huang, C.-J., Katoh, F., Kaneko, T., Hwang, P.-P., 2007. Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio). Am. J. Physiol. -Reg. I. 292, 2068-2076.
21 Hoshijima, K., Hirose, S., 2007. Expression of endocrine genes in zebrafish larvae in response to environmental salinity. J. Endocrinol. 193, 481-491. Hwang, P.-P., 2009. Ion uptake and acid secretion in zebrafish (Danio rerio). J. Exp. Biol. 212, 1745-1752. Hwang, P.-P., Perry, S.F., 2010. Ionic and Acid-Base Regulation, in: S.F. Perry, M. Ekker, A.P. Farrell, C.J. Brauner (Eds.), Zebrafish. Academic Press, 311-344. Hwang, P.P., Chou, M.Y., 2013. Zebrafish as an animal model to study ion homeostasis. Pflugers Arch. 465, 1233-1247. Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D., Peterson, R.T., Yeh, J.R.J., Joung, J.K., 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotech 31, 227-229. Jonz, M.G., Nurse, C.A., 2006. Epithelial mitochondria-rich cells and associated innervation in adult and developing zebrafish. J. Comp. Neurol. 497, 817-832. Kaiya, H., Takei, Y., 1996. Changes in Plasma Atrial and Ventricular Natriuretic Peptide Concentrations after Transfer of Eels from Freshwater to Seawater or Vice Versa. General and Comparative Endocrinology 104, 337-345. Kleszczynska, A., Vargas-Chacoff, L., Gozdowska, M., Kalamarz, H., Martinez-Rodriguez, G., Mancera, J.M., Kulczykowska, E., 2006. Arginine vasotocin, isotocin and melatonin responses following acclimation of gilthead sea bream (Sparus aurata) to different environmental salinities. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 145, 268-273.
22 Kobayashi, H., Uemura, H., Takei, Y., Itatsu, N., Ozawa, M., Ichinohe, K., 1983. Drinking induced by angiotensin II in fishes. Gen. Comp. Endocrinol. 49, 295-306. Kolosov, D., Bui, P., Chasiotis, H., Kelly, S.P., 2013. Claudins in teleost fishes. Tissue Barriers 1, e25391. Kulczykowska, E., 1997. Response of circulating arginine vasotocin and isotocin to rapid osmotic challenge in rainbow trout. Comp. Biochem. Physiol. -A 118, 773-778. Kumai, Y., Bahubeshi, A., Steele, S., Perry, S.F., 2011. Strategies for maintaining Na+ balance in zebrafish (Danio rerio) during prolonged exposure to acidic water. Comp. Biochem. Physiol. -A 160, 52-62. Kumai, Y., Bernier, N.J., Perry, S.F., 2014a. Angiotensin-II promotes Na+ uptake in larval zebrafish, Danio rerio, in acidic and ion-poor water. J. Endocrinol. 220, 195-205. Kumai, Y., Kwong, R.W.M., Perry, S.F., 2014b. A role for transcription factor glial cell missing 2 in Ca2+ homeostasis in zebrafish, Danio rerio. Pflugers Arch - Eur J Physiol 467, 753-765. Kumai, Y., Nesan, D., Vijayan, M.M., Perry, S.F., 2012a. Cortisol regulates Na+ uptake in zebrafish, Danio rerio, larvae via the glucocorticoid receptor. Mol. Cell. Endocrinol. 364, 113125. Kumai, Y., Perry, S.F., 2011. Ammonia excretion via Rhcg1 facilitates Na+ uptake in larval zebrafish, Danio rerio, in acidic water. Am. J. Physiol. - Reg. I. 301, R1517-R1528. Kumai, Y., Porteus, C., Kwong, R.M., Perry, S., 2014c. Hydrogen sulfide inhibits Na+ uptake in larval zebrafish, Danio rerio. Pflugers Arch., 1-14.
23 Kumai, Y., Ward, M.A.R., Perry, S.F., 2012b. β-Adrenergic regulation of Na+ uptake by larval zebrafish Danio rerio in acidic and ion-poor environments. Am. J. Physiol. -Reg. I. 303, R1031R1041. Kwong, R.W., Kumai, Y., Perry, S.F., 2014a. The physiology of fish at low pH: the zebrafish as a model system. J. Exp. Biol. 217, 651-662. Kwong, R.W., Perry, S.F., 2015a. An essential role for parathyroid hormone in gill formation and differentiation of ion-transporting cells in developing zebrafish. Endocrinology 156, 23842394. Kwong, R.W.M., Auprix, D., Perry, S.F., 2014b. Involvement of the calcium-sensing receptor in calcium homeostasis in larval zebrafish exposed to low environmental calcium. Am. J. Physiol. Reg. I. 306, R211-R221. Kwong, R.W.M., Kumai, Y., Perry, S.F., 2013. Evidence for a role of tight junctions in regulating sodium permeability in zebrafish (Danio rerio) acclimated to ion-poor water. J. Comp. Physiol. -B 183, 203-213. Kwong, R.W.M., Perry, S.F., 2013a. Cortisol regulates epithelial permeability and sodium losses in zebrafish exposed to acidic water. J. Endocrinol. 217, 253-264. Kwong, R.W.M., Perry, S.F., 2013b. The tight junction protein claudin-b regulates epithelial permeability and sodium handling in larval zebrafish, Danio rerio. Am. J. Physiol. -Reg. I. 304, R504-R513.
24 Kwong, R.W.M., Perry, S.F., 2015b. Hydrogen sulfide promotes calcium uptake in larval zebrafish. Am J Physiol - Cell Physiol 309, 60-69. Lafont, A.-G., Wang, Y.-F., Chen, G.-D., Liao, B.-K., Tseng, Y.-C., Huang, C.-J., Hwang, P.-P., 2011. Involvement of calcitonin and its receptor in the control of calcium-regulating genes and calcium homeostasis in zebrafish (Danio rerio). J. Bone Miner. Res. 26, 1072-1083. Lee, Z.W., Zhou, J., Chen, C.S., Zhao, Y., Tan, C.H., Li, L., Moore, P.K., Deng, L.W., 2011. The slow-releasing Hydrogen Sulfide donor, GYY4137, exhibits novel anti-cancer effects in vitro and in vivo. PLoS ONE 6. Liao, B.-K., Chen, R.-D., Hwang, P.-P., 2009. Expression regulation of Na+-K+-ATPase α1subunit subtypes in zebrafish gill ionocytes. Am. J. Physiol. -Reg. I. 296, R1897-R1906. Lin, C.-H., Hu, H.-J., Hwang, P.-P., 2016. Cortisol regulates sodium homeostasis by stimulating the transcription of sodium-chloride transporter (NCC) in zebrafish (Danio rerio). Mol. Cell. Endocrinol. 422, 93-102. Lin, C.-H., Shih, T.-H., Liu, S.-T., Hsu, H.-H., Hwang, P.-P., 2015. Cortisol Regulates Acid Secretion of H(+)-ATPase-rich Ionocytes in Zebrafish (Danio rerio) Embryos. Frontiers in Physiology 6, 328. Lin, C.-H., Su, C.-H., Tseng, D.-Y., Ding, F.-C., Hwang, P.-P., 2012. Action of vitamin D and the receptor, VDRa, in calcium handling in zebrafish (Danio rerio). PLoS ONE 7, e45650.
25 Lin, C.-H., Tsai, I.L., Su, C.-H., Tseng, D.-Y., Hwang, P.-P., 2011. Reverse effect of mammalian hypocalcemic cortisol in fish: Cortisol stimulates Ca2+ uptake via glucocorticoid receptormediated vitamin D3 metabolism. PLoS ONE 6, e23689. Lin, C.H., Su, C.H., Hwang, P.P., 2014. Calcium-sensing receptor mediates Ca2+ homeostasis by modulating expression of PTH and stanniocalcin. Endocrinology 155, 56-67. Liu, Y.-W., 2007. Interrenal organogenesis in the zebrafish model. Organogenesis 3, 44-48. Loretz, C.A., Takei, Y., 1997. Natriuretic peptide inhibition of intestinal salt absorption in the Japanese eel: physiological significance. Fish Physiol. Biochem. 17, 319-324. Lukert, B.P., Raisz, L.G., 1990. Glucocorticoid-induced osteoporosis: pathogenesis and management. Ann. Intern. Med. 112, 352-364. Marshall, W.S., Bryson, S.E., Garg, D., 1993. Alpha 2-adrenergic inhibition of Cl- transport by opercular epithelium is mediated by intracellular Ca2+. Proceedings of the National Academy of Sciences 90, 5504-5508. Marsigliante, S., Muscella, A., Vinson, G.P., Storelli, C., 1997. Angiotensin II receptors in the gill of sea water- and freshwater-adapted eel. J. Mol. Endocrinol. 18, 67-76. McDonald, D.G., Tang, Y., Boutilier, R.G., 1989. Acid and ion transfer across the gills of fish: mechanisms and regulation. Canadian Journal of Zoology 67, 3046-3054. Mommsen, T.P., Vijayan, M.M., Moon, T.W., 1999. Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation. Rev. Fish Biol. Fish. 9, 211-268.
26 Mustafa, A.K., Gadalla, M.M., Snyder, S.H., 2009. Signaling by gasotransmitters. Science signaling 2, re2-re2. Okabe, M., Graham, A., 2004. The origin of the parathyroid gland. Proc. Natl. Acad. Sci. USA 101, 17716-17719. Pan, T.C., Liao, B.K., Huang, C.J., Lin, L.Y., Hwang, P.P., 2005. Epithelial Ca2+ channel expression and Ca2+ uptake in developing zebrafish. Am. J. Physiol. -Reg. I. 289, R1202-1211. Perry, S.F., Payan, P., Girard, J.P., 1984. Adrenergic control of branchial chloride transport in the isolated perfused head of the freshwater trout (Salmo gairdneri). Journal of Comparative Physiology B 154, 269-274. Perry, S.F., Reid, S.G., Wankiewicz, E., Iyer, V., Gilmour, K.M., 1996. Physiological responses of rainbow trout (Oncorhynchus mykiss) to prolonged exposure to soft water. Physiological Zoology 69, 1419-1441. Perry, S.I., Vermette, M.G., 1987. The effects of prolonged epinephrine infusion on the physiology of the rainbow trout, Salmo gairdneri. I. Blood respiratory, acid-base and ionic states. J. Exp. Biol. 128, 235-253. Reid, S.G., Bernier, N.J., Perry, S.F., 1998. The adrenergic stress response in fish: control of catecholamine storage and release. Comp. Biochem. Physiol. -C 120, 1-27. Rubin, D.A., Jüppner, H., 1999. Zebrafish express the common parathyroid hormone/parathyroid hormone-related peptide receptor (PTH1R) and a novel receptor (PTH3R) that is preferentially
27 activated by mammalian and fugufish parathyroid hormone-related peptide. The Journal of biological chemistry 274, 28185-28190. Shih, T.-H., Horng, J.-L., Liu, S.-T., Hwang, P.-P., Lin, L.-Y., 2012. Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water. Am. J. Physiol. -Reg. I. 302, 84-93. Shono, T., Kurokawa, D., Miyake, T., Okabe, M., 2011. Acquisition of glial cells missing 2 enhancers contributes to a diversity of ionocytes in zebrafish. PLoS ONE 6, e23746. Shu, Y., Lou, Q., Dai, Z., Dai, X., He, J., Hu, W., Yin, Z., 2016. The basal function of teleost prolactin as a key regulator on ion uptake identified with zebrafish knockout models. Sci. Rep. 6, 18597. Smith, N.F., Eddy, F.B., Struthers, A.D., Talbot, C., 1991. Renin, Atrial Natriuretic Peptide and Blood Plasma Ions in Parr and Smolts of Atlantic Salmon Salmo Salar L. and Rainbow Trout Oncorhynchus Mykiss (Walbaum) in Fresh Water and After Short-Term Exposure to Sea Water. Journal of Experimental Biology 157, 63-74. Steele, S.L., Ekker, M., Perry, S.F., 2011. Interactive effects of development and hypoxia on catecholamine synthesis and cardiac function in zebrafish (Danio rerio). J. Comp. Physiol. -B 181, 527-538. Tipsmark, C.K., Madsen, S.S., 2003. Regulation of Na+/K+-ATPase activity by nitric oxide in the kidney and gill of the brown trout (Salmo trutta). J. Exp. Biol. 206, 1503-1510.
28 To, T.T., Hahner, S., Nica, G., Rohr, K.B., Hammerschmidt, M., Winkler, C., Allolio, B., 2007. Pituitary-interrenal interaction in zebrafish interrenal organ development. Mol. Endocrinol. 21, 472-485. Tseng, D.-Y., Chou, M.-Y., Tseng, Y.-C., Hsiao, C.-D., Huang, C.-J., Kaneko, T., Hwang, P.-P., 2009. Effects of stanniocalcin 1 on calcium uptake in zebrafish (Danio rerio) embryo. Am. J. Physiol. -Reg. I. 296, R549-R557. Tucker, B., Hepperle, C., Kortschak, D., Rainbird, B., Wells, S., Oates, A.C., Lardelli, M., 2007. Zebrafish Angiotensin II Receptor-like 1a (agtrl1a) is expressed in migrating hypoblast, vasculature, and in multiple embryonic epithelia. Gene Expression Patterns 7, 258-265. Turksen, K., Troy, T.C., 2004. Barriers built on claudins. J. Cell Sci. 117, 2435-2447. Vermette, M.G., Perry, S.F., 1987. The effects of prolonged epinephrine infusion on the physiology of the rainbow trout, Salmo gairdneri. II. Branchial solute fluxes. J. Exp. Biol. 128, 255-267. Wang, Y.-F., Tseng, Y.-C., Yan, J.-J., Hiroi, J., Hwang, P.-P., 2009. Role of SLC12A10.2, a NaCl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio). Am. J. Physiol. -Reg. I. 296, 1650-1660. Yan, Y.L., Bhattacharya, P., He, X.J., Ponugoti, B., Marquardt, B., Layman, J., Grunloh, M., Postlethwait, J.H., Rubin, D.A., 2012. Duplicated zebrafish co-orthologs of parathyroid hormone-related peptide (PTHrP, Pthlh) play different roles in craniofacial skeletogenesis. J. Endocrinol. 214, 421-435.
29 Zhang, J., Piontek, J., Wolburg, H., Piehl, C., Liss, M., Otten, C., Christ, A., Willnow, T.E., Blasig, I.E., Abdelilah-Seyfried, S., 2010. Establishment of a neuroepithelial barrier by claudin5a is essential for zebrafish brain ventricular lumen expansion. Proc. Natl. Acad. Sci. USA 107, 1425-1430. Figure legends Figure 1. (A) Fluorescent immunohistochemistry and confocal microscopy showing the H+ATPase-rich cells (HRCs; blue), Na+/Cl--cotransporter expressing cells (NCCCs; green) and Na+/K+-ATPase-rich cells (NaRCs; red) on the skin of yolk sac in larval zebrafish at 4 days post fertilization (dpf). Image (B) shows a higher magnification of the ionocytes. HRCs were labelled with concanavalin A. NCCCs and NaRCs were labelled with a polyclonal antibody raised against the zebrafish NCC and a monoclonal antibody against the α-subunit of avian Na+-K+-ATPase, respectively. Figure 2. The epithelial Ca2+ channels (ecac)-expressing ionocytes are closely associated with an extensive network of nerves. Double in situ hybridization and immunohistochemistry images of larval zebrafish at 4 days post fertilization (dpf) illustrating that the (A) ecac-expressing ionocytes (arrowheads) are surrounded by (B) an extensive network of nerves (stained with a generic neuronal marker zn-12 monoclonal antibody; green) in the skin of the yolk sac. (C) is a merged image of (A) and (B). Scale bar = 20 µm. Figure 3. A proposed model illustrating the effects of neuroendocrine factors on ion transport pathways in zebrafish ionocytes. Neuroendocrine factors may interact with their receptors in ionocytes and subsequently modulate the function of ion transporters. These factors may also interact with ionocyte progenitors and/or cell-fate transcription factors to regulate
30 differentiation/proliferation of ionocytes. Factors labelled with a question mark (?) indicate that the interactions remain unclear. NHE, Na+/H+ exchanger; HA, H+-ATPase; NCC, Na+/Cl-cotransporter; ECaC, epithelial Ca2+ channel; HRC, H+-ATPase-rich cell; NCCC, NCCexpressing cell; NaRC, Na+/K+-ATPase-rich cell; EDN1, endothelin-1; PRL, prolactin; ANG-II, angiotensin-II; STC1, stanniocalcin-1; CT, calcitonin; PTH1, parathyroid hormone-1.
31 Figure 1 A
HRCs NCCCs NaRCs
B
100 µm
HRCs NCCCs NaRCs
50 µm
32 Figure 2 A
B
C
ecac
zn12
merge
33 Figure 3
Water Na+
Na+ Cl-
Stimulation
Ca2+
Inhibition
NHE
Catecholamines H2S (?)
NCC
EDN1 Cortisol Isotocin
Cortisol Isotocin Catecholamines
PRL Cortisol ANG-II (?) Isotocin
(acting via β-receptor)
HRC (+) Cortisol (+) Isotocin (-) STC1
Blood
ECaC
H+
H+
(acting via α-receptor)
HA
NCCC (+) Cortisol (+) Isotocin (+) PRL (-) STC1
STC1 CT
Cortisol PTH1 Isotocin Vitamin D H2S
NaRC (+) Cortisol (+) Isotocin (-) STC1
Factors affecting the number of ionocytes (+) increase (-) decrease
34 Highlights •
Zebrafish is a useful model for studying ion-regulation in vertebrates
•
The neuroendocrine system is critically involved in maintaining ionic homeostasis
•
The neuroendocrine system regulates ion uptake, efflux and ionocytes differentiation
•
The neuroendocrine system is activated to promote ionic balance when zebrafish are exposed to environmental stressors
S0016-6480(16)30137-X http://dx.doi.org/10.1016/j.ygcen.2016.05.016 YGCEN 12404
To appear in:
General and Comparative Endocrinology
Received Date: Revised Date: Accepted Date:
4 October 2015 6 May 2016 11 May 2016
Please cite this article as: Kwong, R.W.M., Kumai, Y., Perry, S.F., Neuroendocrine control of ionic balance in zebrafish, General and Comparative Endocrinology (2016), doi: http://dx.doi.org/10.1016/j.ygcen.2016.05.016
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1
Neuroendocrine control of ionic balance in zebrafish Raymond W.M. Kwong*, Yusuke Kumai1, Steve F. Perry
Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5
*Corresponding author: Raymond W.M. Kwong, Ph.D. Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5 Tel. 1-613-562-5800 ext 6010 E-mail: [email protected]
1
Dr. Kumai passed away suddenly in August 2015
2 Abstract Zebrafish (Danio rerio) is an emerging model for integrative physiological research. In this minireview, we discuss recent advances in the neuroendocrine control of ionic balance in this species, and identify current knowledge gaps and issues that would benefit from further investigation. Zebrafish inhabit a hypo-ionic environment and therefore are challenged by a continual loss of ions to the water. To maintain ionic homeostasis, they must actively take up ions from the water and reduce passive ion loss. The adult gill or the skin of larvae are the primary sites of ionic regulation. Current models for the uptake of major ions in zebrafish incorporate at least three types of ion transporting cells (also called ionocytes); H+-ATPase-rich cells for Na+ uptake, Na+/K+-ATPase-rich cells for Ca2+ uptake, and Na+/Cl--cotransporter expressing cells for both Na+ and Cl- uptake. The precise molecular mechanisms regulating the paracellular loss of ions remain largely unknown. However, epithelial tight junction proteins, including claudins, are thought to play a critical role in reducing ion losses to the surrounding water. Using the zebrafish model, several key neuroendocrine factors were identified as regulators of epithelial ion movement, including the catecholamines (adrenaline and noradrenaline), cortisol, the reninangiotensin system, parathyroid hormone and prolactin. Increasing evidence also suggests that gasotransmitters, such as H2S, are involved in regulating ion uptake.
Keywords: Calcium; cortisol; ionic regulation; ionocyte; neuroendocrine control; sodium
3 1. Introduction Previous studies using traditional models [e.g. rainbow trout (Oncorhynchus mykiss), goldfish (Carassius auratus), killifish (Fundulus heteroclitus) and American eel (Anguilla rostrata)] have provided important information on the fundamental mechanisms of osmoregulation in fish. However, limitations in the use of molecular physiological approaches in these species have hindered precise characterization of the underlying molecular pathways and mechanisms. The zebrafish (Danio rerio) has emerged as an important model for integrative physiological research, owing to the availability of genetic databases, applicability of genetic editing and manipulation, and transparency of the embryos allowing direct cellular/tissue observations. The ion transport pathways in zebrafish are thought to resemble those occurring in different segments of the mammalian kidney (Hwang and Chou, 2013). For example, the expression of different transporters for the absorption of Na+, Ca2+ and Cl-, and their regulatory functions, appear to be conserved between zebrafish gills/skin and mammalian kidney. Thus, the zebrafish is a useful model system for advancing our understanding of the ion-regulatory functions in the mammalian kidney. Several previous studies using zebrafish have identified key neuroendocrine factors which are important in regulating ionic homeostasis and transepithelial ion movements. The regulation of ion movement in zebrafish was described in several previous reviews (Hwang, 2009; Hwang and Perry, 2010; Hwang and Chou, 2013; Kwong et al., 2014a). The present review provides a brief overview of the mechanisms of ionic regulation in zebrafish and discusses some of the recent advances surrounding their neuroendocrine control. More specifically, we focus on the adrenergic and glucocorticoid systems, angiotensin II, parathyroid hormone (PTH), prolactin and gasotransmitters. Finally, this review reveals current knowledge gaps and identifies issues that could be addressed in future studies.
4 2. Molecular physiology of ionic regulation in zebrafish Zebrafish, a freshwater teleost, are hyper-ionic to their environment and thus are challenged constantly by diffusive ion losses. To maintain whole body ionic homeostasis, they need to actively absorb ions from, and reduce passive ion losses to, the environment. In adults, the gills are the predominant site for active ion uptake through specific ion-transporting cells termed ionocytes. During larval stages before the gills are fully developed, regulation of epithelial ion transport is primarily mediated by the ionocytes found on the skin of the yolk sac. Although ionocytes are recognized as the universal site of ionic uptake in fish, there is no common nomenclature that yet has been developed to designate their structure and function. Indeed, the development of a common nomenclature is challenging because of interspecific differences in ionocyte subtype that are known to exist across species (for a review, see Dymowska et al., 2012). Current models for the absorption of the major ions (i.e. Na+, Ca2+ and Cl-) in zebrafish incorporate at least 3 types of ionocytes; H+-ATPase-rich cells (HRCs), Na+/K+-ATPase-rich cells (NaRCs) and Na+/Cl--cotransporter-expressing cells (NCCCs) (Figure 1). HRCs express H+-ATPase and Na+/H+-exchanger at the apical membrane (i.e. NHE3b) for H+ secretion and Na+ uptake, respectively; NCCCs express Na+/Cl--cotransporter for both Na+ and Cl- uptake; a subset of NaRCs express epithelial Ca2+ channel (ECaC) at the apical membrane for Ca2+ uptake, and Na+/Ca2+-exchanger and Ca2+-ATPase at the basolateral membrane for Ca2+ extrusion. Different paralogues of Na+/K+-ATPase are expressed at the basolateral membrane in all these 3 types of ionocytes (Horng et al., 2007; Kumai and Perry, 2011; Liao et al., 2009; Pan et al., 2005; Shih et al., 2012; Wang et al., 2009). Recently, the acid-sensing ion channels (ASICs), the closest relatives of the mammalian epithelial Na+ channels (ENaC), were identified at the apical membrane of HRCs in adult zebrafish gills (Dymowska et al., 2015).
5 Paracellular movement of ions and fluid is regulated predominantly by the tight junction proteins. Occludin, claudins, tricellulins and junctional adhesion molecule are major components of the tight junctions. However, claudins are thought to be the primary determinant of the epithelial barrier functions in vertebrates (Turksen and Troy, 2004). To date, there are over 50 different isoforms of claudins that have been identified in zebrafish, and most of them are expressed in a cell- or tissue-specific manner (Baltzegar et al., 2013; Clelland and Kelly, 2010; Kumai et al., 2011; Kwong and Perry, 2013b). The functional characteristics of most of the claudins remain largely unknown. Nonetheless, certain claudin isoforms appear to form selective pores or barriers to regulate the paracellular movement of ions or fluid (Bagnat et al., 2007; Kwong et al., 2013; Kwong and Perry, 2013b; Zhang et al., 2010), and are critical for development and osmoregulation (for a review, see Kolosov et al., 2013). 3. Neuroendocrine control of ionic regulation in zebrafish 3.1 Catecholamines In vertebrates, the catecholamines, adrenaline and noradrenaline, are essential for regulating cardiovascular and respiratory functions. It is also documented that the adrenergic system is involved in osmoregulation and acid-base balance in fish (Donald, 1989; Kumai et al., 2012b; Marshall et al., 1993; McDonald et al., 1989; Perry et al., 1984; Perry et al., 1996; Perry and Vermette, 1987; Reid et al., 1998; Vermette and Perry, 1987). Catecholamines either can interact with target cells after their secretion as neurotransmitters from nearby nerve endings, or after their release from chromaffin cells for more widespread distribution via the circulatory system. Using high-performance liquid chromatography (HPLC), catecholamines were detectable in whole embryos at 1 h post fertilization (hpf), presumably derived from maternal transfer (Steele et al., 2011). At 2 days post fertilization (dpf), chromaffin cells were found to be
6 dispersed as clusters in the inter-renal organ, which then converged to midline by 3 dpf; the chromaffin cells stay in intimate contact with steroidogenic cells throughout the subsequent development (Liu, 2007; To et al., 2007). In zebrafish, both HRCs and NaRCs are innervated (Jonz and Nurse, 2006; Kumai et al., 2012b). Using fluorescently labeled propranolol (nonselective β-adrenergic receptor antagonist), β receptors were found to be distributed in HRCs, as well as in mitochondrion-rich ionocytes (likely NaRCs). The ecac-expressing NaRCs also are closely associated with an extensive network of nerves (Figure 2). These observations suggest the possible direct control of ionocyte functions by the neuronal systems in zebrafish. Using pharmacological approaches, it was demonstrated that blockade of β-adrenergic receptors decreased Na+ uptake in larval zebrafish, whereas blockade of α-adrenergic receptors increased Na+ uptake (Kumai et al., 2012b). Similarly, pharmacological inhibition of β- and αadrenergic receptors decreased and increased Ca2+ uptake, respectively (R.W.M Kwong and S.F. Perry; unpublished results). Therefore, it appears that β- and α-adrenergic systems play opposing roles in regulating Na+ and Ca2+ uptake. More importantly, knockdown of specific β-adrenergic receptors was shown to inhibit Na+ uptake in larval zebrafish exposed to acidic or ion-poor water, conditions known to increase Na+ transport capacity in this species (Kumai et al., 2012b). These observations suggest that the increased capacity for Na+ uptake is, at least in part, mediated by activation of the β-adrenergic system. Interestingly, Na+ uptake appears to be regulated by different β receptors under different environmental conditions (i.e. β1 or β2b receptors are involved in increasing Na+ uptake during exposure to acidic water, while β2a receptors play a role in ion-poor conditions) (Kumai et al., 2012b). However, the precise molecular pathways underlying the increased Na+ uptake and the intracellular signalling cascade are yet to be fully
7 characterized. The specific role of different subtypes of β- or α-receptors in modulating ion uptake also requires further investigation. 3.2 Cortisol Cortisol is involved in regulating a diverse range of biological processes in fish, including reproduction, metabolism, immune responses and osmoregulation (Mommsen et al., 1999). Similar to some other freshwater species, exposure of zebrafish to cortisol stimulates the uptake of both Na+ and Ca2+ (Kumai et al., 2012a; Lin et al., 2016; Lin et al., 2011), and the secretion of H+ (Lin et al., 2015). Using in situ hybridization or immunohistochemistry, the glucocorticoid receptor (GR) was found to be expressed in NaRCs (Cruz et al., 2013), HRCs (Kumai et al., 2012a) and NCCCs (Lin et al., 2016) in zebrafish. Exposure to cortisol was also shown to increase mRNA expression of genes that are involved in ion uptake or H+ secretion, including ecac, ncc, nhe3b, atp6v1a, ae1b, ca2-like a, and ca15a (Lin et al., 2015; Lin et al., 2011). Additionally, morpholino knockdown of the GR prevented the increase in mRNA expression of ecac and ncc caused by cortisol treatment (Lin et al., 2016; Lin et al., 2011). Cortisol also modulates the mRNA expression of enzymes/hormone known to regulate Ca2+ uptake, including reducing the hypocalcemic hormone stanniocalcin (stc-1) (Kumai et al., 2014b) and increasing several enzymes responsible for vitamin-D synthesis (Lin et al., 2012). Furthermore, it was demonstrated that cortisol treatment stimulated the mRNA expression of glial cell missing-2 (gcm2) (Kumai et al., 2014b) and forkhead box I3 (foxi3a and foxi3b) (Cruz et al., 2013a), which are transcription factors critically involved in ionocyte development in zebrafish (Chang et al., 2009; Kumai et al., 2014b; Shono et al., 2011). Similarly, GR knockdown was shown to decrease the numbers of HRCs and NaRCs (Cruz et al., 2013b).
8 Together, these studies clearly demonstrate that cortisol plays an important role in promoting ion uptake and ionocyte differentiation in zebrafish. In zebrafish, cortisol appears to be involved in promoting ionic balance during exposure to environmental stressors. For example, the mRNA expression of GR was found to increase in larval zebrafish acclimated to softwater (i.e. low Ca2+ water) (Lin et al., 2011). The mRNA levels of 11β-hydroxylase, a vital enzyme responsible for cortisol synthesis, also was increased in zebrafish acclimated to softwater (Lin et al., 2011). Additionally, whole body cortisol levels in zebrafish were elevated following exposure to low Na+ (Lin et al., 2016) or acidic (pH 4.0) water (Kumai et al., 2012a). In acidic water, the elevated cortisol levels appear to promote Na+ balance by increasing transcellular Na+ uptake as well as by reducing paracellular Na+ losses, probably via activation of the cortisol-GR signalling cascade (Kumai et al., 2012a; Kwong and Perry, 2013a). Cortisol likely stimulates Na+ uptake through the NHE3b–Rhcg1 functional metabolon (Kumai et al., 2012a). The exact mechanisms for the reduction in paracellular Na+ loss by cortisol are not fully understood, but appear to be associated with an increase in the abundance of epithelial tight junction proteins (Kwong and Perry, 2013a). Overall, these findings demonstrate that the cortisol-GR signalling cascade is involved in promoting ionic balance in zebrafish. Interestingly, cortisol is reported to act as a hypocalcemic factor in mammals (Lukert and Raisz, 1990), suggesting different roles for cortisol in ionic regulation in mammals and zebrafish. In other fish species, cortisol is reported to interact with other hormonal systems known to modulate osmoregulation, such as growth hormone and insulin-like growth factor (Mommsen et al., 1999); whether similar interactions also occur in zebrafish remain unclear, and would be an important area for future investigation. 3.3 Angiotensin II
9 In mammals, the renin–angiotensin system (RAS) is involved in regulating salt reabsorption in the kidney (Crowley and Coffman, 2012). Angiotensinogen is first cleaved to angiotensin-I (ANG-I) by the enzyme renin, which is converted to the biologically active form ANG-II by angiotensin-converting enzyme (ACE). The physiological role of the RAS in fish is not completely understood. Nevertheless, increasing evidence suggests that the RAS is involved in osmoregulation (Fuentes and Eddy, 1997; Hoshijima and Hirose, 2007; Kumai et al., 2014a; Smith et al., 1991). It is well documented that ANG-II promotes drinking in euryhaline fish (Beasley et al., 1986; Carrick and Balment, 1983; Fuentes and Eddy, 1996; Kobayashi et al., 1983), potentially increasing salt absorption from the gut. In the European eel Anguilla anguilla, receptors for ANG-II are located in gill ionocytes, and treatment of eels with ANG-II caused an increase in the activity of Na+/K+-ATPase (Marsigliante et al., 1997). In larval zebrafish, ANG-II receptor-like 1a (agtrl1a) is expressed in multiple tissues, including developing lens, otic vesicles and venous vasculature (Tucker et al., 2007). It is unknown whether ANG-II receptors are expressed in ionocytes of zebrafish larvae or adults. Previous studies have provided evidence that the RAS promotes ion uptake in zebrafish. For example, acclimation of larval zebrafish to ion-poor water increased the mRNA expression of renin (Hoshijima and Hirose, 2007). Additionally, whole body ANG-II levels were elevated in zebrafish exposed to ion-poor or acidic water (Kumai et al., 2014a). Importantly, translational gene knockdown of renin substantially attenuated the increased Na+ uptake following acute exposure to acidic or ion-poor water (Kumai et al., 2014a), indicating a role for the RAS in promoting Na+ influx. The downstream signalling pathways in the acute regulation of Na+ uptake by the RAS remain unknown. In fish, ANG-II is a potent stimulant for cortisol synthesis (Arnold-Reed and Balment, 1994), and cortisol is known to promote Na+ uptake in larval
10 zebrafish (discussed above). Interestingly, the increase in Na+ uptake associated with acute acid or ion-poor water exposure was not affected by GR knockdown in larval zebrafish, implying cortisol is not involved in RAS-mediated Na+ regulation (Kumai et al., 2014a). The precise Na+transport pathways which are regulated by the RAS also remain unclear. However, treatment with exogenous ANG-II was found to increase the mRNA expression of NCC (Kumai et al., 2014a). Although ANG-II appears to play a role in stimulating Na+ uptake, the identification of ANG-II receptors in ionocytes or in osmoregulatory tissues, and the underlying molecular mechanisms, have yet to be investigated in zebrafish. 3.4 Parathyroid hormone In mammals, PTH is expressed and secreted predominantly by the parathyroid gland. PTH plays an important role in regulating Ca2+ metabolism and skeletal remodeling. Fish do not have parathyroid glands, and the gill has been proposed to be the evolutionary precursor of the mammalian parathyroid glands (Okabe and Graham, 2004). Zebrafish express two PTH paralogs, PTH1 and PTH2 (Hogan et al., 2005). Their mRNA expression have been detected in various tissues in adults (Lin et al., 2014), and primarily in the central nervous system in larvae (Hogan et al., 2005). Three PTH receptors (PTH1R-a, PTH1R-b and PTH2R) have been identified in zebrafish (Hoare et al., 2000; Rubin and Jüppner, 1999), which appear to be expressed in a tissue-specific manner in adults (Kwong and Perry, 2015a). Similar to mammals, PTH functions as a hypercalcemic hormone and regulates skeletogenesis in zebrafish. To date, no PTH receptor has been identified in zebrafish ionocytes. However, acclimation of larval zebrafish to ion-poor or low Ca2+ water was shown to increase the mRNA expression of pth1 (Hoshijima and Hirose, 2007; Kwong and Perry, 2015a; Lin et al.,
11 2014). Such modulation is likely mediated via the extracellular Ca2+-sensing receptor expressed in ionocytes and/or corpuscles of Stannius (Kwong et al., 2014b; Lin et al., 2014). Additionally, translational gene knockdown of PTH1 reduced whole body Ca2+ levels and decreased mRNA expression of ecac in larval zebrafish (Kwong and Perry, 2015a; Lin et al., 2014). PTH1 also appears to be important in promoting ionocyte differentiation and cartilage development, probably through its interaction with the cell-fate transcription factor gcm2 (Kwong and Perry, 2015a). Furthermore, overexpression of the hypocalcemic hormone calcitonin was found to increase the mRNA expression of PTH receptors in zebrafish (Lafont et al., 2011), suggesting a compensatory role of PTH in maintaining Ca2+ balance. In some other fish species, the PTHrelated protein (PTHrP; a protein member of the PTH family) also was suggested to be an essential hypercalcaemic factor (Abbink and Flik, 2007). Similar to PTH, PTHrP is reported to be critical for skeletogenesis in developing zebrafish (Yan et al., 2012). Whether PTHrP also regulates Ca2+ acquisition in zebrafish remains to be determined. 3.5 Prolactin Several previous studies have suggested that prolactin is important in ionic regulation in zebrafish. In larval zebrafish, prolactin is expressed in the anterior pituitary (Liu et al., 2006; Sbrogna et al., 2003), and its receptors (prlra and prlrb) are abundantly expressed in both the developing gills and kidney (Shu et al., 2016). In adult zebrafish, these receptors are expressed in osmoregulatory organs, including the gills, kidney and intestine (Breves et al., 2013b). It was demonstrated that larval zebrafish exposed to dilute freshwater exhibited an increase in the mRNA expression of prolactin (Hoshijima and Hirose, 2007). Intraperitoneal injection of ovine prolactin increased mRNA expression of ncc and prolactin receptor-a (prlra) in adult zebrafish gills (Breves et al., 2013a). Results from whole-mount in situ hybridization also revealed that the
12 number of ncc-positive cells was reduced in larval zebrafish experiencing NCC knockdown (Breves et al., 2014). Using a line of prolactin-knockout mutants, it was further demonstrated that prolactindeficient larvae could not survive to the adult stage when they were raised in typical freshwater (2 mOsm/L) (Shu et al., 2016). The prolactin-deficient larvae at 6 dpf had lower whole body levels of Na+, K+ and Cl− when compared to the wild-type fish (Shu et al., 2016). Interestingly, the reduction in whole body ion levels in the prolactin-deficient zebrafish was prevented when they were raised in ion-enriched water (163 mOsm/L), allowing them to survive into adults (Shu et al., 2016). Additionally, a decrease in the mRNA expression levels of ncc but increased expression levels of atp1a1a.5 (an HRC marker) was observed in prolactin-deficient zebrafish larvae (Shu et al., 2016). Overall, these studies demonstrate an important role of prolactin in maintaining ionic balance and survival during early development, at least in part via its regulation of the expression of NCC. However, whether prolactin regulates NCC expression directly, or differentiation of NCCCs from ionocyte progenitors is unclear. Future studies should address the underlying mechanisms linking prolactin and ionocyte development and the role of prolactin in regulating NCC function. Prolactin is proposed to be a freshwater-adapting hormone not only by promoting ion uptake, but also by reducing water permeability (Sakamoto and McCormick, 2006). The zebrafish model may prove useful in elucidating the potential role of prolactin in regulating epithelial permeability and the associated downstream signalling processes. 3.6 Gasotransmitters
13 Gasotransmitters, including nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S), are endogenously produced gaseous signalling molecules which regulate a variety of physiological functions in vertebrates, including the cardiovascular, respiratory, nervous and immune systems (Mustafa et al., 2009). Increasing evidence suggests that gasotransmitters may play a role in osmoregulation in fish (Ebbesson et al., 2005; Kumai et al., 2014c; Kwong and Perry, 2015b; Tipsmark and Madsen, 2003). To date, only the role of H2S in ionic regulation has been investigated in zebrafish. Biosynthesis of H2S is predominantly mediated by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), and in adult zebrafish, mRNA expression of cbsb and cse are detected in various tissues, including the gill, intestine and kidney (Kwong and Perry, 2015b). Both CBSb and CSE are expressed in HRCs and NaRCs in larval zebrafish (Kumai et al., 2014c; Kwong and Perry, 2015b), suggesting a capacity of these ionocytes to produce H2S. It was demonstrated that exposure to exogenous H2S donors, either Na2S or GYY4137 (a slow-releasing H2S donor; Lee et al., 2011) substantially reduced Na+ uptake in larval zebrafish (Kumai et al., 2014c). Using pharmacological agents or genetic knockdown approaches, it was shown that inhibition of the H2S-synthesising enzymes significantly attenuated the capacity of fish to lower their Na+ uptake after exposure to Na+-enriched water (Kumai et al., 2014c). These findings indicate that H2S is a physiologically relevant signaling molecule which contributes to reducing Na+ uptake capacity in developing zebrafish. The precise molecular targets for H2S have not been fully characterized. However, treatment with H2S donors did not reduce Na+ uptake in fish lacking HRCs (Kumai et al., 2014c), suggesting H2S interacts with HRCs in reducing Na+ uptake. Interestingly, as opposed to its inhibitory role in Na+ uptake, H2S appears to stimulate Ca2+ uptake in larval zebrafish. For example, exposure to H2S donors Na2S or GYY4137
14 increased Ca2+ influx and whole body Ca2+ content in larval zebrafish (Kwong and Perry, 2015b). Additionally, gene knockdown of CBSb significantly reduced Ca2+ influx in larval zebrafish acclimated to a low Ca2+ environment (Kwong and Perry, 2015b). The mRNA expression of cbsb was also found to increase in fish acclimated to a low Ca2+ environment (Kwong and Perry, 2015b). The exact mechanisms for the stimulatory effects of H2S on Ca2+ influx are not clear, but appear to involve post-translational activation of ECaC via the cAMP-PKA pathways (Kwong and Perry, 2015b). The significance of H2S in reducing Na+ influx while simultaneously increasing Ca2+ influx is unknown. However, these observations suggest that H2S regulates Na+ and Ca2+ uptake through different mechanisms in different cell types. To fully address the physiological relevance of H2S, future studies should examine the levels of endogenous H2S and/or activity of H2S-producing enzymes in response to different environmental conditions, and the effects of H2S on different ion channels/transporters in the various ionocyte subtypes. The potential role of other gasotransmitters (e.g. NO and CO) in ionic regulation, the precise molecular targets for gasotransmitters, and the intracellular signalling pathways, are worthwhile areas for future research. 3.7 Other neuroendocrine factors Several other hormones, such as endothelin, stanniocalcin, isotocin and atrial natriuretic peptide (ANP) are suggested to be involved in regulating ion uptake, ionocyte development and/or acidbase balance in zebrafish. For example, endothelin and stanniocalcin were found to play a critical role in stimulating H+ secretion (Guh et al., 2014) and reducing Ca2+ uptake (Tseng et al., 2009), respectively. In adult zebrafish, isotocin mRNA is predominantly expressed in the brain but also is expressed in other organs including the gill, muscle and ovaries (Chou et al., 2011). Receptors for isotocin (both itnpr-like 1 and itnpr-like 2) are ubiquitously expressed in adults tissues (Chou
15 et al., 2011). In rainbow trout Oncorhynchus mykiss (Kulczykowska, 1997) and sea bream Sparus aurata (Kleszczynska et al., 2006), isotocin levels increase with increasing salinity, suggesting its possible role in hyperosmotic acclimation. Interestingly, mRNA levels of isotocin in larval zebrafish were significantly higher following their exposure to deionised water (Chou et al., 2011). Additionally, gene knockdown of isotocin substantially reduced the whole-body levels of Na+, Ca2+ and Cl- (Chou et al., 2011). Isotocin also appears to be important in promoting the development of various ionocytes, probably through its interaction with epidermal stem cells and foxi3a-expressing ionocyte progenitors (Chou et al., 2011). These findings suggest that in zebrafish, isotocin plays a critical role in promoting ion retention and ionocyte development. A substantial body of evidence suggests that ANP is a seawater-adapting hormone in fish. For example, in the Japanese eel Anguilla japonica, plasma levels of ANP increase after transferring from freshwater to seawater, and treatment with ANP inhibited intestinal Na+ uptake (Kaiya and Takei, 1996; Loretz and Takei, 1997). In larval zebrafish, the expression of anp was confined to the atrium (Berdougo et al., 2003). Interestingly, anp expression was found to substantially increase in larvae following exposure to ion-poor water (Hoshijima and Hirose, 2007). Clearly, the functional role of ANP in zebrafish osmoregulation is virtually unknown and warrants further study. 4. Conclusions and perspectives The zebrafish has emerged as a powerful model vertebrate to study the neuroendocrine control of ionic regulation. Several key neuroendocrine factors and their downstream regulatory pathways have been elucidated using the zebrafish model (Figure 3). These findings have provided new insights into the molecular physiology of ionic regulation in fish. However, the response of these
16 neuroendocrine factors to environmental challenges (e.g. ion-poor water), with a few exceptions (cortisol, ANG II), are largely unknown, and should be addressed in future research. Currently, reverse genetics approaches, particularly the morpholino technology, are used routinely in larval zebrafish to investigate ion regulatory functions. However, the morpholino technique only allows transient gene knockdown during early developmental stages, and is not suitable for assessing gene function in adults. Moreover, there are obvious physiological differences in the ionregulatory systems between larval and adult fish (e.g. ion regulation occurs primarily at the gill, intestine and kidney in adults, whereas it occurs in the skin of yolk sac in larvae). Therefore, larvae and adults may employ different strategies to maintain ionic balance. It is important that future studies examine the regulation of ionic balance (as well as other physiological functions) in adult animals. Recent advances on the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems (Hwang et al., 2013) will undoubtedly prove useful in assessing mechanisms of ion regulation in adults. Despite the obvious advantages, there are also some significant limitations associated with using zebrafish as a model for piscine osmoregulation. For example, the relatively small size and lack of euryhalinity in zebrafish hinder certain experimental procedures, such as evaluation of blood chemistry and effects of osmotic stress. Thus, a continuing comparative approach aimed at assessing inter-specific similarities and differences in ion-regulatory mechanisms arguably is the preferred approach to shed light on the diverse strategies employed by fish to achieve ionic and osmotic homeostasis. 5. Acknowledgements
17 Our original research was financially supported by Natural Sciences and Engineering Research Council (NSERC) Discovery and NSERC Research Tools and Instrumentation Grants to S.F. Perry.
18 Reference Abbink, W., Flik, G., 2007. Parathyroid hormone-related protein in teleost fish. Gen. Comp. Endocrinol. 152, 243-251. Arnold-Reed, D.E., Balment, R.J., 1994. Peptide Hormones Influence in Vitro Interrenal Secretion of Cortisol in the Trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 96, 85-91. Bagnat, M., Cheung, I.D., Mostov, K.E., Stainier, D.Y.R., 2007. Genetic control of single lumen formation in the zebrafish gut. Nat. Cell Biol. 9, 954-960. Baltzegar, D.A., Reading, B.J., Brune, E.S., Borski, R.J., 2013. Phylogenetic revision of the claudin gene family. Marine Genomics 11, 17-26. Beasley, D., Shier, D.N., Malvin, R.L., Smith, G., 1986. Angiotensin-stimulated drinking in marine fish. Am. J. Physiol. 250, R1034-1038. Berdougo, E., Coleman, H., Lee, D.H., Stainier, D.Y.R., Yelon, D., 2003. Mutation of weak atrium/atrial myosin heavy chain disrupts atrial function and influences ventricular morphogenesis in zebrafish. Development 130, 6121-6129. Carrick, S., Balment, R.J., 1983. The renin-angiotensin system and drinking in the euryhaline flounder, Platichthys flesus. Gen. Comp. Endocrinol. 51, 423-433. Chang, W.J., Horng, J.L., Yan, J.J., Hsiao, C.D., Hwang, P.P., 2009. The transcription factor, glial cell missing 2, is involved in differentiation and functional regulation of H+-ATPase-rich cells in zebrafish (Danio rerio). Am. J. Physiol. -Reg. I. 296, R1192-R1201.
19 Chou, M.-Y., Hung, J.-C., Wu, L.-C., Hwang, S.-P., Hwang, P.-P., 2011. Isotocin controls ion regulation through regulating ionocyte progenitor differentiation and proliferation. Cell. Mol. Life Sci. 68, 2797-2809. Clelland, E.S., Kelly, S.P., 2010. Tight junction proteins in zebrafish ovarian follicles: Stage specific mRNA abundance and response to 17β-estradiol, human chorionic gonadotropin, and maturation inducing hormone. Gen. Comp. Endocrinol. 168, 388-400. Crowley, S.D., Coffman, T.M., 2012. Recent advances involving the renin–angiotensin system. Exp. Cell Res. 318, 1049-1056. Cruz, S.A., Chao, P.-L., Hwang, P.-P., 2013a. Cortisol promotes differentiation of epidermal ionocytes through Foxi3 transcription factors in zebrafish (Danio rerio). Comp. Biochem. Physiol. -A 164, 249-257. Cruz, S.A., Lin, C.-H., Chao, P.-L., Hwang, P.-P., 2013b. Glucocorticoid receptor, but not mineralocorticoid Receptor, mediates cortisol regulation of epidermal Ionocyte development and Ion transport in zebrafish (Danio Rerio). PLoS ONE 8, e77997. Donald, J.A., 1989. Adrenaline and branchial nerve stimulation inhibit 45Ca influx into the gills of rainbow trout, Salmo Gairdneri. Journal of Experimental Biology 141, 441-445. Dymowska, A.K., Boyle, D., Schultz, A.G., Goss, G.G., 2015. The role of acid-sensing ion channels (ASICs) in epithelial Na+ uptake in adult zebrafish (Danio rerio). J. Exp. Biol. Dymowska, A.K., Hwang, P.-P., Goss, G.G., 2012. Structure and function of ionocytes in the freshwater fish gill. Res. Physiol. Neurobiol. 184, 282-292.
20 Ebbesson, L.O., Tipsmark, C.K., Holmqvist, B., Nilsen, T., Andersson, E., Stefansson, S.O., Madsen, S.S., 2005. Nitric oxide synthase in the gill of Atlantic salmon: colocalization with and inhibition of Na+,K+-ATPase. J. Exp. Biol. 208, 1011-1017. Fuentes, J., Eddy, F.B., 1996. Drinking in Freshwater-Adapted Rainbow Trout Fry, Oncorhynchus mykiss (Walbaum), in Response to Angiotensin I, Angiotensin II, AngiotensinConverting Enzyme Inhibition, and Receptor Blockade. Physiological Zoology 69, 1555-1569. Fuentes, J., Eddy, F.B., 1997. Drinking in marine, euryhaline and freshwater teleost fish, in: N. Hazon, F.B. Eddy, G. Flik (Eds.), Ionic Regulation in Animals: A Tribute to Professor W.T.W.Potts. Springer Berlin Heidelberg, 135-149. Guh, Y.J., Tseng, Y.C., Yang, C.Y., Hwang, P.P., 2014. Endothelin-1 regulates H+-ATPasedependent transepithelial H+ secretion in zebrafish. Endocrinology 155, 1728-1737. Hoare, S.R.J., Rubin, D.A., Jüppner, H., Usdin, T.B., 2000. Evaluating the ligand specificity of zebrafish parathyroid hormone (PTH) receptors: Comparison of PTH, PTH-Related protein, and tuberoinfundibular peptide of 39 residues. Endocrinology 141, 3080-3086. Hogan, B.M., Danks, J.A., Layton, J.E., Hall, N.E., Heath, J.K., Lieschke, G.J., 2005. Duplicate zebrafish pth genes are expressed along the lateral line and in the central nervous system during embryogenesis. Endocrinology 146, 547-551. Horng, J.-L., Lin, L.-Y., Huang, C.-J., Katoh, F., Kaneko, T., Hwang, P.-P., 2007. Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio). Am. J. Physiol. -Reg. I. 292, 2068-2076.
21 Hoshijima, K., Hirose, S., 2007. Expression of endocrine genes in zebrafish larvae in response to environmental salinity. J. Endocrinol. 193, 481-491. Hwang, P.-P., 2009. Ion uptake and acid secretion in zebrafish (Danio rerio). J. Exp. Biol. 212, 1745-1752. Hwang, P.-P., Perry, S.F., 2010. Ionic and Acid-Base Regulation, in: S.F. Perry, M. Ekker, A.P. Farrell, C.J. Brauner (Eds.), Zebrafish. Academic Press, 311-344. Hwang, P.P., Chou, M.Y., 2013. Zebrafish as an animal model to study ion homeostasis. Pflugers Arch. 465, 1233-1247. Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D., Peterson, R.T., Yeh, J.R.J., Joung, J.K., 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotech 31, 227-229. Jonz, M.G., Nurse, C.A., 2006. Epithelial mitochondria-rich cells and associated innervation in adult and developing zebrafish. J. Comp. Neurol. 497, 817-832. Kaiya, H., Takei, Y., 1996. Changes in Plasma Atrial and Ventricular Natriuretic Peptide Concentrations after Transfer of Eels from Freshwater to Seawater or Vice Versa. General and Comparative Endocrinology 104, 337-345. Kleszczynska, A., Vargas-Chacoff, L., Gozdowska, M., Kalamarz, H., Martinez-Rodriguez, G., Mancera, J.M., Kulczykowska, E., 2006. Arginine vasotocin, isotocin and melatonin responses following acclimation of gilthead sea bream (Sparus aurata) to different environmental salinities. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 145, 268-273.
22 Kobayashi, H., Uemura, H., Takei, Y., Itatsu, N., Ozawa, M., Ichinohe, K., 1983. Drinking induced by angiotensin II in fishes. Gen. Comp. Endocrinol. 49, 295-306. Kolosov, D., Bui, P., Chasiotis, H., Kelly, S.P., 2013. Claudins in teleost fishes. Tissue Barriers 1, e25391. Kulczykowska, E., 1997. Response of circulating arginine vasotocin and isotocin to rapid osmotic challenge in rainbow trout. Comp. Biochem. Physiol. -A 118, 773-778. Kumai, Y., Bahubeshi, A., Steele, S., Perry, S.F., 2011. Strategies for maintaining Na+ balance in zebrafish (Danio rerio) during prolonged exposure to acidic water. Comp. Biochem. Physiol. -A 160, 52-62. Kumai, Y., Bernier, N.J., Perry, S.F., 2014a. Angiotensin-II promotes Na+ uptake in larval zebrafish, Danio rerio, in acidic and ion-poor water. J. Endocrinol. 220, 195-205. Kumai, Y., Kwong, R.W.M., Perry, S.F., 2014b. A role for transcription factor glial cell missing 2 in Ca2+ homeostasis in zebrafish, Danio rerio. Pflugers Arch - Eur J Physiol 467, 753-765. Kumai, Y., Nesan, D., Vijayan, M.M., Perry, S.F., 2012a. Cortisol regulates Na+ uptake in zebrafish, Danio rerio, larvae via the glucocorticoid receptor. Mol. Cell. Endocrinol. 364, 113125. Kumai, Y., Perry, S.F., 2011. Ammonia excretion via Rhcg1 facilitates Na+ uptake in larval zebrafish, Danio rerio, in acidic water. Am. J. Physiol. - Reg. I. 301, R1517-R1528. Kumai, Y., Porteus, C., Kwong, R.M., Perry, S., 2014c. Hydrogen sulfide inhibits Na+ uptake in larval zebrafish, Danio rerio. Pflugers Arch., 1-14.
23 Kumai, Y., Ward, M.A.R., Perry, S.F., 2012b. β-Adrenergic regulation of Na+ uptake by larval zebrafish Danio rerio in acidic and ion-poor environments. Am. J. Physiol. -Reg. I. 303, R1031R1041. Kwong, R.W., Kumai, Y., Perry, S.F., 2014a. The physiology of fish at low pH: the zebrafish as a model system. J. Exp. Biol. 217, 651-662. Kwong, R.W., Perry, S.F., 2015a. An essential role for parathyroid hormone in gill formation and differentiation of ion-transporting cells in developing zebrafish. Endocrinology 156, 23842394. Kwong, R.W.M., Auprix, D., Perry, S.F., 2014b. Involvement of the calcium-sensing receptor in calcium homeostasis in larval zebrafish exposed to low environmental calcium. Am. J. Physiol. Reg. I. 306, R211-R221. Kwong, R.W.M., Kumai, Y., Perry, S.F., 2013. Evidence for a role of tight junctions in regulating sodium permeability in zebrafish (Danio rerio) acclimated to ion-poor water. J. Comp. Physiol. -B 183, 203-213. Kwong, R.W.M., Perry, S.F., 2013a. Cortisol regulates epithelial permeability and sodium losses in zebrafish exposed to acidic water. J. Endocrinol. 217, 253-264. Kwong, R.W.M., Perry, S.F., 2013b. The tight junction protein claudin-b regulates epithelial permeability and sodium handling in larval zebrafish, Danio rerio. Am. J. Physiol. -Reg. I. 304, R504-R513.
24 Kwong, R.W.M., Perry, S.F., 2015b. Hydrogen sulfide promotes calcium uptake in larval zebrafish. Am J Physiol - Cell Physiol 309, 60-69. Lafont, A.-G., Wang, Y.-F., Chen, G.-D., Liao, B.-K., Tseng, Y.-C., Huang, C.-J., Hwang, P.-P., 2011. Involvement of calcitonin and its receptor in the control of calcium-regulating genes and calcium homeostasis in zebrafish (Danio rerio). J. Bone Miner. Res. 26, 1072-1083. Lee, Z.W., Zhou, J., Chen, C.S., Zhao, Y., Tan, C.H., Li, L., Moore, P.K., Deng, L.W., 2011. The slow-releasing Hydrogen Sulfide donor, GYY4137, exhibits novel anti-cancer effects in vitro and in vivo. PLoS ONE 6. Liao, B.-K., Chen, R.-D., Hwang, P.-P., 2009. Expression regulation of Na+-K+-ATPase α1subunit subtypes in zebrafish gill ionocytes. Am. J. Physiol. -Reg. I. 296, R1897-R1906. Lin, C.-H., Hu, H.-J., Hwang, P.-P., 2016. Cortisol regulates sodium homeostasis by stimulating the transcription of sodium-chloride transporter (NCC) in zebrafish (Danio rerio). Mol. Cell. Endocrinol. 422, 93-102. Lin, C.-H., Shih, T.-H., Liu, S.-T., Hsu, H.-H., Hwang, P.-P., 2015. Cortisol Regulates Acid Secretion of H(+)-ATPase-rich Ionocytes in Zebrafish (Danio rerio) Embryos. Frontiers in Physiology 6, 328. Lin, C.-H., Su, C.-H., Tseng, D.-Y., Ding, F.-C., Hwang, P.-P., 2012. Action of vitamin D and the receptor, VDRa, in calcium handling in zebrafish (Danio rerio). PLoS ONE 7, e45650.
25 Lin, C.-H., Tsai, I.L., Su, C.-H., Tseng, D.-Y., Hwang, P.-P., 2011. Reverse effect of mammalian hypocalcemic cortisol in fish: Cortisol stimulates Ca2+ uptake via glucocorticoid receptormediated vitamin D3 metabolism. PLoS ONE 6, e23689. Lin, C.H., Su, C.H., Hwang, P.P., 2014. Calcium-sensing receptor mediates Ca2+ homeostasis by modulating expression of PTH and stanniocalcin. Endocrinology 155, 56-67. Liu, Y.-W., 2007. Interrenal organogenesis in the zebrafish model. Organogenesis 3, 44-48. Loretz, C.A., Takei, Y., 1997. Natriuretic peptide inhibition of intestinal salt absorption in the Japanese eel: physiological significance. Fish Physiol. Biochem. 17, 319-324. Lukert, B.P., Raisz, L.G., 1990. Glucocorticoid-induced osteoporosis: pathogenesis and management. Ann. Intern. Med. 112, 352-364. Marshall, W.S., Bryson, S.E., Garg, D., 1993. Alpha 2-adrenergic inhibition of Cl- transport by opercular epithelium is mediated by intracellular Ca2+. Proceedings of the National Academy of Sciences 90, 5504-5508. Marsigliante, S., Muscella, A., Vinson, G.P., Storelli, C., 1997. Angiotensin II receptors in the gill of sea water- and freshwater-adapted eel. J. Mol. Endocrinol. 18, 67-76. McDonald, D.G., Tang, Y., Boutilier, R.G., 1989. Acid and ion transfer across the gills of fish: mechanisms and regulation. Canadian Journal of Zoology 67, 3046-3054. Mommsen, T.P., Vijayan, M.M., Moon, T.W., 1999. Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation. Rev. Fish Biol. Fish. 9, 211-268.
26 Mustafa, A.K., Gadalla, M.M., Snyder, S.H., 2009. Signaling by gasotransmitters. Science signaling 2, re2-re2. Okabe, M., Graham, A., 2004. The origin of the parathyroid gland. Proc. Natl. Acad. Sci. USA 101, 17716-17719. Pan, T.C., Liao, B.K., Huang, C.J., Lin, L.Y., Hwang, P.P., 2005. Epithelial Ca2+ channel expression and Ca2+ uptake in developing zebrafish. Am. J. Physiol. -Reg. I. 289, R1202-1211. Perry, S.F., Payan, P., Girard, J.P., 1984. Adrenergic control of branchial chloride transport in the isolated perfused head of the freshwater trout (Salmo gairdneri). Journal of Comparative Physiology B 154, 269-274. Perry, S.F., Reid, S.G., Wankiewicz, E., Iyer, V., Gilmour, K.M., 1996. Physiological responses of rainbow trout (Oncorhynchus mykiss) to prolonged exposure to soft water. Physiological Zoology 69, 1419-1441. Perry, S.I., Vermette, M.G., 1987. The effects of prolonged epinephrine infusion on the physiology of the rainbow trout, Salmo gairdneri. I. Blood respiratory, acid-base and ionic states. J. Exp. Biol. 128, 235-253. Reid, S.G., Bernier, N.J., Perry, S.F., 1998. The adrenergic stress response in fish: control of catecholamine storage and release. Comp. Biochem. Physiol. -C 120, 1-27. Rubin, D.A., Jüppner, H., 1999. Zebrafish express the common parathyroid hormone/parathyroid hormone-related peptide receptor (PTH1R) and a novel receptor (PTH3R) that is preferentially
27 activated by mammalian and fugufish parathyroid hormone-related peptide. The Journal of biological chemistry 274, 28185-28190. Shih, T.-H., Horng, J.-L., Liu, S.-T., Hwang, P.-P., Lin, L.-Y., 2012. Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water. Am. J. Physiol. -Reg. I. 302, 84-93. Shono, T., Kurokawa, D., Miyake, T., Okabe, M., 2011. Acquisition of glial cells missing 2 enhancers contributes to a diversity of ionocytes in zebrafish. PLoS ONE 6, e23746. Shu, Y., Lou, Q., Dai, Z., Dai, X., He, J., Hu, W., Yin, Z., 2016. The basal function of teleost prolactin as a key regulator on ion uptake identified with zebrafish knockout models. Sci. Rep. 6, 18597. Smith, N.F., Eddy, F.B., Struthers, A.D., Talbot, C., 1991. Renin, Atrial Natriuretic Peptide and Blood Plasma Ions in Parr and Smolts of Atlantic Salmon Salmo Salar L. and Rainbow Trout Oncorhynchus Mykiss (Walbaum) in Fresh Water and After Short-Term Exposure to Sea Water. Journal of Experimental Biology 157, 63-74. Steele, S.L., Ekker, M., Perry, S.F., 2011. Interactive effects of development and hypoxia on catecholamine synthesis and cardiac function in zebrafish (Danio rerio). J. Comp. Physiol. -B 181, 527-538. Tipsmark, C.K., Madsen, S.S., 2003. Regulation of Na+/K+-ATPase activity by nitric oxide in the kidney and gill of the brown trout (Salmo trutta). J. Exp. Biol. 206, 1503-1510.
28 To, T.T., Hahner, S., Nica, G., Rohr, K.B., Hammerschmidt, M., Winkler, C., Allolio, B., 2007. Pituitary-interrenal interaction in zebrafish interrenal organ development. Mol. Endocrinol. 21, 472-485. Tseng, D.-Y., Chou, M.-Y., Tseng, Y.-C., Hsiao, C.-D., Huang, C.-J., Kaneko, T., Hwang, P.-P., 2009. Effects of stanniocalcin 1 on calcium uptake in zebrafish (Danio rerio) embryo. Am. J. Physiol. -Reg. I. 296, R549-R557. Tucker, B., Hepperle, C., Kortschak, D., Rainbird, B., Wells, S., Oates, A.C., Lardelli, M., 2007. Zebrafish Angiotensin II Receptor-like 1a (agtrl1a) is expressed in migrating hypoblast, vasculature, and in multiple embryonic epithelia. Gene Expression Patterns 7, 258-265. Turksen, K., Troy, T.C., 2004. Barriers built on claudins. J. Cell Sci. 117, 2435-2447. Vermette, M.G., Perry, S.F., 1987. The effects of prolonged epinephrine infusion on the physiology of the rainbow trout, Salmo gairdneri. II. Branchial solute fluxes. J. Exp. Biol. 128, 255-267. Wang, Y.-F., Tseng, Y.-C., Yan, J.-J., Hiroi, J., Hwang, P.-P., 2009. Role of SLC12A10.2, a NaCl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio). Am. J. Physiol. -Reg. I. 296, 1650-1660. Yan, Y.L., Bhattacharya, P., He, X.J., Ponugoti, B., Marquardt, B., Layman, J., Grunloh, M., Postlethwait, J.H., Rubin, D.A., 2012. Duplicated zebrafish co-orthologs of parathyroid hormone-related peptide (PTHrP, Pthlh) play different roles in craniofacial skeletogenesis. J. Endocrinol. 214, 421-435.
29 Zhang, J., Piontek, J., Wolburg, H., Piehl, C., Liss, M., Otten, C., Christ, A., Willnow, T.E., Blasig, I.E., Abdelilah-Seyfried, S., 2010. Establishment of a neuroepithelial barrier by claudin5a is essential for zebrafish brain ventricular lumen expansion. Proc. Natl. Acad. Sci. USA 107, 1425-1430. Figure legends Figure 1. (A) Fluorescent immunohistochemistry and confocal microscopy showing the H+ATPase-rich cells (HRCs; blue), Na+/Cl--cotransporter expressing cells (NCCCs; green) and Na+/K+-ATPase-rich cells (NaRCs; red) on the skin of yolk sac in larval zebrafish at 4 days post fertilization (dpf). Image (B) shows a higher magnification of the ionocytes. HRCs were labelled with concanavalin A. NCCCs and NaRCs were labelled with a polyclonal antibody raised against the zebrafish NCC and a monoclonal antibody against the α-subunit of avian Na+-K+-ATPase, respectively. Figure 2. The epithelial Ca2+ channels (ecac)-expressing ionocytes are closely associated with an extensive network of nerves. Double in situ hybridization and immunohistochemistry images of larval zebrafish at 4 days post fertilization (dpf) illustrating that the (A) ecac-expressing ionocytes (arrowheads) are surrounded by (B) an extensive network of nerves (stained with a generic neuronal marker zn-12 monoclonal antibody; green) in the skin of the yolk sac. (C) is a merged image of (A) and (B). Scale bar = 20 µm. Figure 3. A proposed model illustrating the effects of neuroendocrine factors on ion transport pathways in zebrafish ionocytes. Neuroendocrine factors may interact with their receptors in ionocytes and subsequently modulate the function of ion transporters. These factors may also interact with ionocyte progenitors and/or cell-fate transcription factors to regulate
30 differentiation/proliferation of ionocytes. Factors labelled with a question mark (?) indicate that the interactions remain unclear. NHE, Na+/H+ exchanger; HA, H+-ATPase; NCC, Na+/Cl-cotransporter; ECaC, epithelial Ca2+ channel; HRC, H+-ATPase-rich cell; NCCC, NCCexpressing cell; NaRC, Na+/K+-ATPase-rich cell; EDN1, endothelin-1; PRL, prolactin; ANG-II, angiotensin-II; STC1, stanniocalcin-1; CT, calcitonin; PTH1, parathyroid hormone-1.
31 Figure 1 A
HRCs NCCCs NaRCs
B
100 µm
HRCs NCCCs NaRCs
50 µm
32 Figure 2 A
B
C
ecac
zn12
merge
33 Figure 3
Water Na+
Na+ Cl-
Stimulation
Ca2+
Inhibition
NHE
Catecholamines H2S (?)
NCC
EDN1 Cortisol Isotocin
Cortisol Isotocin Catecholamines
PRL Cortisol ANG-II (?) Isotocin
(acting via β-receptor)
HRC (+) Cortisol (+) Isotocin (-) STC1
Blood
ECaC
H+
H+
(acting via α-receptor)
HA
NCCC (+) Cortisol (+) Isotocin (+) PRL (-) STC1
STC1 CT
Cortisol PTH1 Isotocin Vitamin D H2S
NaRC (+) Cortisol (+) Isotocin (-) STC1
Factors affecting the number of ionocytes (+) increase (-) decrease
34 Highlights •
Zebrafish is a useful model for studying ion-regulation in vertebrates
•
The neuroendocrine system is critically involved in maintaining ionic homeostasis
•
The neuroendocrine system regulates ion uptake, efflux and ionocytes differentiation
•
The neuroendocrine system is activated to promote ionic balance when zebrafish are exposed to environmental stressors