Tight junctions, tight junction proteins and paracellular permeability across the gill epithelium of fishes: A review

Respiratory Physiology & Neurobiology 184 (2012) 269–281

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Review

Tight junctions, tight junction proteins and paracellular permeability across the gill epithelium of fishes: A review夽 Helen Chasiotis, Dennis Kolosov, Phuong Bui, Scott P. Kelly ∗ Department of Biology, York University, Toronto, ON, Canada M3J 1P3

a r t i c l e

i n f o

Article history: Accepted 20 May 2012 Keywords: Gill Occludin Claudin ZO-1 Pavement cell Mitochondrion-rich cell

a b s t r a c t Paracellular permeability characteristics of the fish gill epithelium are broadly accepted to play a key role in piscine salt and water balance. This is typically associated with differences between gill epithelia of teleost fishes residing in seawater versus those in freshwater. In the former, the gill is ‘leaky’ to facilitate Na+ secretion and in the latter, the gill is ‘tight’ to limit passive ion loss. However, studies in freshwater fishes also suggest that varying epithelial ‘tightness’ can impact ionoregulatory homeostasis. Paracellular permeability of vertebrate epithelia is largely controlled by the tight junction (TJ) complex, and the fish gill is no exception. In turn, the TJ complex is composed of TJ proteins, the abundance and properties of which determine the magnitude of paracellular solute movement. This review provides consolidated information on TJs in fish gills and summarizes recent progress in research that seeks to understand the molecular composition of fish gill TJ complexes and what environmental and systemic factors influence those components. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The gill is an architecturally complex organ that occupies a central role in the physiology of fishes (for review see, Evans et al., 2005). The parenchyma of gill is a heterogeneous epithelium which is directly exposed to a surrounding environment of water. The gill epithelium presents a large surface area for the movement of biological material between blood and water, and the gill is simultaneously involved in respiration, iono/osmoregulation, acid-base balance and waste nitrogen excretion (see Evans et al., 2005). The majority of gill epithelial cells that interface with water (>90%) are squamous (or in some cases cuboidal), polygonal pavement cells (PVCs) (Wilson and Laurent, 2002; Fig. 1a). In addition, cuboidal or columnar mitochondrion-rich gill ionocytes (mitochondrion-rich cells, MRCs) are also exposed to water via their apical surface (see Wilson and Laurent, 2002). The apical surfaces of MRCs emerge through the ‘cellular carpet’ of PVCs (Fig. 1b) and are focal points of transcellular ion transport (see Evans et al., 2005; Hwang et al., 2011; Marshall, 2002; Perry, 1997). Because the ionic composition of fish blood is almost always different from that of surrounding

夽 This paper is part of a special issue entitled “New Insights into Structure/Function Relationships in Fish Gills”, guest-edited by William K. Milsom and Steven F. Perry. ∗ Corresponding author at: Department of Biology, York University, 4700 Keele Street, Toronto, ON, Canada M3J 1P3. Tel.: +1 416 736 2100x77830; fax: +1 416 736 5698. E-mail address: [email protected] (S.P. Kelly). 1569-9048/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.resp.2012.05.020

water, the transcellular transport of solutes across the gill epithelium contributes significantly to the overall maintenance of salt and water balance in fishes. However, by virtue of its sizeable surface area the gill is also believed to play a prominent role in obligatory solute movement (loss or gain), much of which is thought to occur across the paracellular pathway between gill epithelial cells (Evans et al., 2005; Marshall and Grosell, 2006). The regulation of solute movement across the paracellular pathway of the fish gill epithelium is generally linked to the properties of the gill tight junction (TJ) complex. In line with the cellular heterogeneity of the gill epithelium, gill TJs are also known to be heterogeneous, most often in association with differences in environmental salt content (Wilson and Laurent, 2002). In this regard, fish gill TJs are broadly acknowledged to play an appreciable role in the maintenance of electrochemical/concentration gradients between fish blood and surrounding water, either by limiting or facilitating paracellular ion movement (see Marshall and Grosell, 2006). Yet until recently, relatively few studies have made the gill TJ complex and/or its molecular components a focal point of study. So in contrast to the well studied transcellular transport characteristics of fish gill epithelia (see Evans et al., 2005; Hwang et al., 2011; Marshall, 2002; Perry, 1997), less is known about the paracellular pathway of the fish gill and the proteins that may contribute to its physiological properties. Nevertheless, mounting curiosity in the sphere of vertebrate TJs has led to a flourish of research interest in fish gill TJ physiology. Therefore, this review seeks to consolidate what is classically known about gill TJs with new insights into the molecular physiology of the gill TJ complex.

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Fig. 1. Gill morphology of spotted green puffer fish (Tetraodon nigroviridis) following acclimation to seawater (SW). Representative electron micrographs show (a) general surface ultrastructure and (b) a higher magnification image of gill pavement cells (PVCs, denoted by asterisks) with the apical crypts of gill mitochondrion-rich cells (MRCs, denoted by arrow heads) interspersed between. In panel (c) the apical region of a gill MRC can be seen in section, with a gill accessory cell (AC) located adjacent. Panel (d) shows a shallow ‘leaky’ gill TJ linking a gill MRC and AC. These ‘leaky’ TJs are only found in teleosts residing in SW. In contrast, TJs that link other cells such as (e) PVC-MRC and (f) PVC-PVC are deep and ‘tight’. The depth of TJs between gill cells in (d–f) is indicated by a black arrow. SL, secondary lamellae. In (a) scale bar = 20 ␮m, (b) 5 ␮m, (c) 1 ␮m and (d–f) 200 nm.

2. The vertebrate tight junction complex

3. Tight junctions in the gills of fishes

In the archetypal tripartite junctional complex of vertebrate epithelia, the zonula occludens or tight junction (TJ) is the outermost juxtaluminal element (Farquhar and Palade, 1963). The TJ is a beltlike reticular network of anastomosing strands that has both a ‘gate’ and ‘fence’ function in epithelia (see Fig. 2). As a ‘fence’, the TJ complex demarcates the apical and basolateral domain of an epithelial cell and as a ‘gate’, the TJ operates as a diffusion barrier that selectively regulates the passage of solutes between epithelial cells (for review, see González-Mariscal et al., 2003). In this regard, it is the TJ complex that plays a dominant role in regulating the paracellular movement of solutes across vertebrate epithelia (Fig. 2). The TJ complex is composed of transmembrane and cytosolic TJ proteins, the latter of which provides structural support to the complex by linking transmembrane TJ proteins to the actin cytoskeleton of an epithelial cell (Fig. 3, for review, see González-Mariscal et al., 2003). However, it is the transmembrane TJ proteins, such as occludin and members of the claudin superfamily, that bridge the intercellular space between epithelial cells and largely influence TJ complex permeability (see Fig. 3; González-Mariscal et al., 2003; Van Itallie and Anderson, 2006).

3.1. Tight junctions in the gills of Myxini and Petromyzontida TJs have been found in the gill epithelia of every extant class of fish (Mallatt and Paulsen, 1986; Bartels and Potter, 1991; Carmona et al., 2004; Sardet et al., 1979; Wilson et al., 2002; Wright, 1974). This is not unexpected given that branchial TJs appear early in the chordate lineage (Burighel et al., 1992; Georges, 1979; Martinucci et al., 1988), and as noted previously, most fishes have to maintain large ionic gradients between body fluids and surrounding water. The only exception to this occurs in stenohaline marine myxinids (hagfishes). In this group of osmoconforming agnathan fishes, blood serum exhibits an almost identical ionic profile to that of seawater (SW) (Robertson, 1954; Currie and Edwards, 2010), although it is generally considered to be slightly hyperosmotic (Parry, 1966). Despite this, deep TJs are still present between the gill epithelial cells that bridge the two media (Bartels, 1988; Mallatt and Paulsen, 1986; Mallatt et al., 1987). Deep multi-stranded TJs are normally presumed to be physiologically ‘tight’ in vertebrate epithelia (González-Mariscal et al., 2003). However, freeze-fracture analysis has also revealed ‘leaky spots’ in the deep branchial TJs

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Fig. 2. Transepithelial solute movement across vertebrate epithelia. Transcellular solute movement occurs across epithelial cell membranes via membrane-bound pumps, transporters and channels. Paracellular solute movement occurs between epithelial cells along the intercellular space by diffusion. The tight junction (shown in purple) appears as a belt-like network of anastomosing strands. These act as a ‘gate’ to the paracellular pathway by occluding the intercellular space and selectively regulating the passage of solutes between epithelial cells. The tight junction also forms a ‘fence’ that prevents membrane-bound molecules on the apical side of epithelia (red) from intermixing with those on the basolateral side (green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3. Generalized model of the tight junction (TJ) complex between epithelial cells. Tetraspan transmembrane TJ proteins such as occludin (shown in purple) and claudins (shown in red) directly regulate the permselectivity characteristics of the TJ by bridging the intercellular space (via their extracellular domains) to form the TJ barrier. ZO-1 (shown in blue) provides structural support to TJs by linking the transmembrane TJ proteins to the actin cytoskeleton. ZO-1 also participates in intracellular signaling pathways related to gene expression, cell proliferation and differentiation by interacting with cytosolic signaling molecules. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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of hagfish (Bartels, 1988). To the best of our knowledge the paracellular permeability properties of hagfish gill epithelia are still unknown, so the exact nature of the gill TJ complex in this group awaits further attention. In contrast to the myxinids, other extant agnathans such as members of the Petromyzontidae family (lampreys), exhibit basic strategies of osmoregulation that are not unlike those of derived fishes such as teleosts. Specifically, the blood of this agnathan group is hyperosmotic when the animals are in FW, and in species that enter a marine setting as part of an anadromous life cycle, the blood is hyposmotic relative to surrounding SW (Parry, 1966). When in FW, lampreys are reported to possess deep multi-stranded TJs between PVCs, PVCs–MRCs, and MRCs–MRCs (Bartels and Potter, 1993). In contrast, when animals are acclimated to SW, MRC–MRC TJs comprise only a single strand or two parallel strands, and as such are presumed to be ‘leaky’ (Bartels and Potter, 1991). The junctional relationships between PVCs and PVCs–MRCs in SW-acclimated fish remain the same as in FW, i.e. deep and ‘tight’ (Bartels and Potter, 1991). From a TJ standpoint, these morphological observations largely mirror those outlined for teleost fishes (see Section 4). As such, they are also presumed to mirror the functional role of ‘deep’ and ‘shallow’ TJs in the gills of teleost fishes (see Section 4). 3.2. Tight junctions in the gills of Chondrichthyes Very little is known about the characteristics of TJs between gill epithelial cells of chondrichthyans. Almost all of these fishes are marine and it has been reported that TJs between gill PVCs and MRCs in at least one species (Squalus acanthius) are deep and presumably ‘tight’ (Wilson et al., 2002). In addition, gill MRCs in chondrichthyans do not occur adjacent to other mitochondrionrich ionocytes, but instead appear singly and surrounded by PVCs (see Wilson and Laurent, 2002). In this regard, no MRC–MRC junctions have been found in the gill epithelia of this group despite the fact that chondrichthyans are hypoionic to surrounding SW. However, in chondrichthyes it is the rectal gland and kidney that play a dominant role in salt secretion and not the gill (Marshall and Grosell, 2006). Nevertheless, gill TJ morphology in these fishes is reminiscent of myxinids rather than Petromyzontidae and not unlike myxinids, chondrichthyan blood plasma is generally considered to be slightly hyperosmotic to SW due to high circulating levels of organic osmolytes (Parry, 1966). In this regard, the presence of gill MRCs in both myxinids and chondrichthyans has been linked to acid/base regulation, the mechanisms of which do not require movement of equivalents through a paracellular shunt (Mallatt et al., 1987; Wilson and Laurent, 2002). 3.3. Tight junctions in the gills of Sarcopterygii To the best of our knowledge, only one study has reported characteristics of TJ morphology in sarcopterygians (see Wright, 1974). In the gill epithelium of Lepidosiren paradoxa, Wright (1974) described intercellular channels between gill epithelial cells that ended subapically at junctional complexes between surface epithelial cells. It can be presumed that these junctions are ‘tight’ because the body surface, including the gill epithelium, of sarcopterygians has been reported to exhibit low ion permeability (Wilkie et al., 2007). Nevertheless, it would be interesting to find out more about branchial TJs in this group of fishes. 3.4. Tight junctions in the gills of non-teleost Actinopterygii Unlike the aforementioned classes, gill TJs have been characterized to a much greater extent in the Actinopterygii. However, this is the result of work conducted on fishes in the Teleostei infraclass. As such, not all groups within the class Actinopterygii have

been examined equally and to the best of our knowledge, only one report describes gill TJs in a species within the subclass Chondrostei (Acipensar naccarii, Carmona et al., 2004), and no reports are available for other non-teleost Actinopterygii. In the case of the Acipenseriformes, this is surprising given that there are at least 11 anadromous species capable of maintaining ionoregulatory homeostasis in both FW and SW (McDowall, 1988). In A. naccarii, Carmona et al. (2004) reported deep TJs between gill PVCs and between PVCs and MRCs. However Carmona et al. (2004) did not mention whether shallow ‘leaky’ TJs were present in the gills of this species or not. Studies on transcellular gill ion transport mechanisms in anadromous Acipenseriformes suggest that strategies employed by this group are similar to those present in teleosts (e.g. Allen et al., 2009). Therefore, because ‘leaky’ TJs are present in the gills of teleosts residing in SW, it will be interesting to see whether ‘leaky’ TJs are eventually reported in SW dwelling Acipenseriformes. All additional knowledge on the structure and function of fish gill TJs are based on studies using Actinopterygii that are members of the infraclass Teleostei. These will be the focus of the remainder of this review. In addition, where further information is required on the structure and function of fish gill epithelia, several excellent reviews are available (Evans et al., 2005; Hwang et al., 2011; Marshall, 2002; Perry, 1997; Wilson and Laurent, 2002), as well as articles published in this special issue.

4. Tight junctions in teleost fish gills and the influence of environmental salt content Variation in gill epithelium permeability is most often linked to comparisons of the ultrastructural and electrophysiological properties of FW and SW teleost fish gills or their surrogate models (e.g. Foskett et al., 1981; Karnaky, 2001; Sardet et al., 1979). Specifically, the gill epithelium of SW (or SW-acclimated) teleost fishes is generally considered to be ‘leakier’ than the gill epithelium of FW (or FW-acclimated) teleost fishes. Increased permeability across the gill epithelium of fish in SW is attributed to the presence of shallow ‘leaky’ TJs that connect interdigitating gill accessory cells (ACs) with MRCs (see Fig. 1c and d). These junctions facilitate the movement of Na+ down its electrochemical gradient into the apical crypts of SW MRCs (Evans et al., 2005; Hwang and Lee, 2007; Marshall, 2002). Nevertheless, in the reminder of the SW fish gill epithelium, deep TJs link MRCs as well as ACs with adjacent PVCs (Fig. 1e), and PVCs to adjacent PVCs (Fig. 1f) (Sardet et al., 1979), suggesting that the majority of the SW fish gill epithelium is not ‘leaky’. In contrast to SW fish gills, there are no ‘leaky’ TJs in the gill epithelium of FW teleost fishes. This is because interdigitating ACs are reported to be absent in the FW teleost fish gill epithelium (Laurent and Dunel, 1980; Sardet et al., 1979; Wilson and Laurent, 2002) and TJs between all epithelial cells are characterized as being deep and ‘tight’ (Kikuchi, 1977; Sardet et al., 1979). However, it has been reported that TJs between adjacent MRCs (Hwang, 1988) and between MRCs and non-interdigitating presumptive ACs (Pisam et al., 1989) may be shallower than those between other gill cell types in FW fishes. Nevertheless, the paracellular pathway is thought to be relatively impermeable to solute movement in FW fishes and in this regard, the ‘tighter’ gill epithelium is recognized to limit passive ion loss in a hyposmotic environment (for review, see Evans et al., 2005; Hwang and Lee, 2007; Marshall, 2002). Often the broadly acknowledged importance of TJs in FW fish gill paracellular permeability stops here. However, a number of early studies also connected alterations in TJ morphology, physiology, and/or paracellular permeability with changes in the rate of ion loss across branchial epithelia or a surrogate model of the branchial epithelium of FW fishes (Freda et al., 1991; Marshall, 1985; McDonald

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Table 1 Occludin expression in the gill tissue and gill epithelial cells of teleost fishes and factors that are currently known to regulate occludin mRNA and/or protein abundance. Species

Gill expression

Regulation

Refs.

Carassius auratus

Whole tissue PVCs, MRCs PVCs

Food deprivation Salinity, cortisol Salinity Cortisol, fish serum

Chasiotis and Kelly (2008) Chasiotis et al. (2009, 2012) and Chasiotis and Kelly (2012) Chasiotis et al. (2012) Chasiotis et al. (2012) and Chasiotis and Kelly (2011a,b)

Oncorhynchus mykiss

Whole tissue PVCs

Not known Cortisol, Dex, RU486

Chasiotis and Kelly (2011a) and Chasiotis et al. (2010) Chasiotis and Kelly (2011a), Chasiotis et al. (2010) and Kelly and Chasiotis (2011)

Danio rerio Fundulus heteroclitus

Whole tissue Whole tissue

pH Salinity

Clelland and Kelly (2010) and Kumai et al. (2011) Whitehead et al. (2011)

PVCs, pavement cells; MRCs, mitochondrion-rich cells; Dex, Dexamethasone.

et al., 1991). These observations clearly suggested that the generic deep TJs found between FW fish gill epithelial cells may also play a dynamic and important role in FW fish ionoregulation. 5. Tight junction proteins in the gills of teleost fishes Since the discovery of the first TJ protein over 20 years ago, more than 40 TJ-associated transmembrane and cytoplasmic proteins have been identified in vertebrate epithelia (González-Mariscal et al., 2003). These molecular TJ constituents are believed to be responsible for the ‘tightness’ of the TJ barrier as well as TJ permselectivity (i.e. the ability to impede or facilitate paracellular solute movement based on size and charge). However, not all identified TJ proteins contribute to the TJ ‘gate’ function (as described in Section 2). A number of TJ proteins have also been recognized to fulfill a wide range of other functions, such as TJ assembly, and the regulation of gene expression, cell differentiation and cell proliferation (reviewed by González-Mariscal et al., 2003). Nevertheless, with respect to TJ barrier function, transmembrane TJ proteins such as occludin and members of the claudin superfamily, and the cytosolic TJ protein ZO-1 have received the most attention in vertebrate (predominantly mammalian) tissues. In line with this, these TJ proteins have been subject to recent investigations in teleost fish gills and will be the focus of this review. 5.1. Occludin in the gill epithelium of fishes Occludin was the first transmembrane TJ protein identified and isolated from vertebrate epithelia (Furuse et al., 1993). This tetraspan protein localizes exclusively to TJ fibrils (i.e. TJ strands; see Fig. 2) at sites of cell-cell contact and is found in a wide variety of epithelial and endothelial tissues (González-Mariscal et al., 2003). Multiple lines of evidence in mammalian models support a role for occludin in the formation and enhancement of the TJ barrier via its extracellular domains, which “occlude” the intercellular space and thus restrict the paracellular movement of solutes (see Fig. 3; reviewed by Cummins, 2012; González-Mariscal et al., 2003). At present, occludin represents the most comprehensively characterized and best understood TJ protein in teleost fishes (Table 1). Full-length cloning studies in goldfish (Carassius auratus) and rainbow trout (Oncorhynchus mykiss) revealed ∼89% and 76% amino acid sequence similarity respectively with zebrafish (Danio rerio) occludin, and ∼60% similarity with mammalian forms (Chasiotis et al., 2010; Chasiotis and Kelly, 2011b). Expression profiles in fishes have demonstrated widespread occludin mRNA distribution among discrete tissues (Chasiotis et al., 2010; Chasiotis and Kelly, 2011b; Clelland and Kelly, 2010; Kumai et al., 2011). However, gill tissues exhibit the highest levels of occludin transcript abundance, thus suggesting an important role for occludin in the regulation of branchial permeability (Chasiotis et al., 2010; Chasiotis and Kelly, 2011b). Because occludin immunostaining within goldfish gills was detected along the edges of lamellae, as well as in cells that showed

positive immunoreactivity for Na+ –K+ -ATPase, it was proposed that occludin is associated with both PVCs and MRCs of the FW fish gill epithelium (Chasiotis and Kelly, 2008). This was later substantiated by a goldfish gill cell separation study, in which comparable levels of occludin transcript abundance were demonstrated between isolated PVC and MRC fractions (Chasiotis et al., 2012). Occludin immunostaining however was also detected within the vasculature of goldfish gill filaments (Chasiotis and Kelly, 2008). Therefore, when interpreting occludin expression (or abundance) data from whole gill tissue, it should be remembered that contributions from the gill epithelium and vasculature are both present. Acclimation of FW goldfish to ion-poor water (IPW) significantly elevated occludin mRNA and protein abundance in gill tissue (Chasiotis et al., 2009, 2012). Using density gradient isolated gill epithelial cells, it has been confirmed that increased gill occludin abundance in IPW occurs in both PVCs and MRCs (Chasiotis et al., 2012). This was shown to occur in association with increased TJ depth between neighbouring PVCs and PVCs adjacent to MRCs (Chasiotis et al., 2012). Furthermore, using microarray analyses Whitehead et al. (2011) reported that gill occludin transcript abundance was up-regulated in SW populations of Atlantic killifish following hypo-osmotic challenge (Whitehead et al., 2011). Taking the above observations together with reports of significantly reduced Na+ efflux across the gills of both IPW-acclimated goldfish (Cuthbert and Maetz, 1972) and FW-adapted killifish (Scott et al., 2004), these studies collectively suggest that occludin likely contributes to a “tightening” of the gill epithelium. A barrier-forming role for occludin in the teleost gill is in accord with the functional characterization of mammalian occludin (reviewed by Cummins, 2012), and has been confirmed by mechanistic studies utilizing in vitro gill models (see Section 7). Such changes in gill TJ physiology would be expected to benefit fishes in a dilute environment, where passive salt loss across the gill would need to be minimized, and particularly in IPW, where active ion acquisition by the gill is limited. Alterations in gill occludin transcript and/or protein abundance have additionally been reported in fish exposed to low pH water (Kumai et al., 2011), following food deprivation (Chasiotis and Kelly, 2008) and when circulating levels of cortisol are chronically elevated (Chasiotis and Kelly, 2012), indicating that gill occludin may be sensitive to a number of environmental and/or systemic variables. 5.2. Claudins in the gill epithelium of fishes Claudins form a large multi-gene family of tetraspan transmembrane TJ proteins that are expressed in epithelial and endothelial cells in a tissue-specific manner (Krause et al., 2008). Due to the properties of their cell-bridging extracellular domains (see Fig. 3), a considerable body of research has implicated select claudins in the formation of charge-selective channels (or pores) within the mammalian TJ complex (reviewed by Krause et al., 2008). These ‘leaky’ claudins have been shown to augment the paracellular

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permeation of charged solutes, and in particular small ions. In contrast, various other members of the mammalian claudin family have been identified as ‘tightening’ or barrier-building claudins that hinder paracellular solute movement (Krause et al., 2008). These contrasting properties together with: (1) the number of members within the claudin superfamily (i.e. at least 24 proteins identified in mammals), (2) the numerous possible combinations of claudin associations (e.g. homo- and heterotypic) within and between TJ fibrils and, (3) the diversity of claudin permselectivity properties, have resulted in the general belief that claudins bestow the wideranging and distinct paracellular permeability properties exhibited within and between different types of vertebrate epithelia and endothelia (Krause et al., 2008; Van Itallie and Anderson, 2006). In teleost fishes, whole genome duplication as well as tandem gene duplication events have resulted in an expansion of the claudin superfamily. This expansion was meticulously reported by Loh et al. (2004) who used the Japanese puffer fish Fugu (=Takifugu) rubripes genome to provide the most comprehensive account of claudins in fishes to date. Loh et al. (2004) described the characteristics and tissue specific distribution patterns of ∼56 claudin genes in Fugu, and since then, the characteristics and distribution of large numbers of claudins have been reported in other fish models, such as the spotted green puffer fish (Tetraodon nigroviridis; Bagherie-Lachidan et al., 2008, 2009; Bui and Kelly, 2011; Bui et al., 2010), goldfish (C. auratus; Chasiotis and Kelly, 2011a,b), zebrafish (D. rerio, Clelland and Kelly, 2010; Kumai et al., 2011), and Atlantic salmon (Salmo salar; Tipsmark et al., 2008b) (see Table 2). Teleost claudin nomenclature is based largely on the nomenclature of human orthologs (Loh et al., 2004). However, where no human ortholog has been identified, novel fish claudins are either numbered starting from 25 and onwards (e.g. Loh et al., 2004 for Fugu) or assigned a character suffix (Kollmar et al., 2001 for D. rerio) (see Table 2). At present, ∼38% of puffer fish claudin genes have not been described in mammals. While a few claudins appear to be broadly expressed in fish tissues, most members of the expanded claudin family exhibit distinct tissue-specific expression patterns (e.g. see Bagherie-Lachidan et al., 2008, 2009; Chasiotis and Kelly, 2012). In addition, claudin abundance has also been reported to vary spatially within fish tissues (e.g. Chasiotis and Kelly, 2012; Clelland et al., 2010; Tipsmark et al., 2008b). This suggests that despite an increase in numbers, piscine claudins have evolved unique and non-redundant functions. At least 32 claudins are expressed in teleost (puffer fish) gills, and within gill tissue, claudins exhibit differential levels of abundance. It is presumed that highly expressed claudins likely act as important regulators of gill permeability (Bagherie-Lachidan et al., 2008, 2009; Bui and Kelly, 2011; Chasiotis and Kelly, 2011a,b; Chasiotis and Kelly, 2012; Kumai et al., 2011; Loh et al., 2004; Tipsmark et al., 2008b). Based on the ‘tight’ versus ‘leaky’ paradigm that has been proposed to exist between TJs of the FW versus SW teleost gill epithelium (see Section 4), numerous studies that have acclimated euryhaline fishes to hypo- and hyper-osmotic environments have provided valuable insights into the potential role of various claudins in the teleost gill. For example, puffer fish claudin-3a, -3c and -8d mRNA, Atlantic killifish claudin-3 and -4 transcripts, and tilapia and Southern flounder claudin-3-like and -4-like proteins were found to be significantly elevated in the gills of fishes acclimated to FW versus those in SW (Bagherie-Lachidan et al., 2008, 2009; Duffy et al., 2011; Tipsmark et al., 2008a,c; Whitehead et al., 2011). Furthermore, claudin-3a and -3c transcripts in the puffer fish gill were shown to be up-regulated following FW to IPW acclimation, and in association with a significant increase in TJ depth between adjacent gill PVCs (Duffy et al., 2011). Taken together, these observations suggest barrier-forming roles for claudin-3, -4 and -8 orthologs in teleost gills, which is in line with the established

function of corresponding orthologous claudins in mammalian models (e.g. Milatz et al., 2010; Van Itallie et al., 2001; Yu et al., 2003). Some differences however have been noted that may be related to variations between species. For example, transcript encoding for claudin-27a was found to be largely unchanged in the gills of Atlantic salmon acclimated from FW to SW (Tipsmark et al., 2008b), but significantly increased in the gills of puffer fish (T. nigroviridis) acclimated from FW to SW (Bagherie-Lachidan et al., 2009), thus confounding insight into the potential function of this claudin gene. Claudin-30, on the other hand, exhibits a greater degree of consistency between species under different conditions. For example, claudin-30 transcript levels are lower in the gills of SW versus FW-acclimated Atlantic salmon (Tipsmark et al., 2008b), first leading to the proposal that this claudin may have a barrier forming role in the gills of fishes since FW fish gills are ‘tighter’ than SW fish gills. In line with these observations, claudin-30 transcript abundance has been demonstrated to increase in association with reduced gill epithelium permeability in trout (Chasiotis and Kelly, 2011a; Kelly and Chasiotis, 2011) and transcript encoding for the claudin-30 ortholog, claudin-b, was up-regulated in gill tissue, as well as in isolated goldfish gill PVCs and MRCs following acclimation from FW to IPW (Chasiotis et al., 2012). This ‘tightening’ role for claudin-30 was confirmed by retroviral transduction of salmon claudin-30 into Madin–Darby canine kidney (MDCK) cells, where a reduction in epithelial conductance and a decrease in permeability to monovalent cations were observed (Engelund et al., 2012). Claudin-30 was also localized to Atlantic salmon gill filament and lamellar surfaces, but showed limited co-localization with Na+ –K+ -ATPase, thus suggesting a greater association with gill PVCs than MRCs (Engelund et al., 2012). These observations support those of Chasiotis and Kelly (2011a), who reported goldfish claudin-30 ortholog (claudin-b) transcript to be very abundant in primary cultured goldfish gill PVCs. Indeed, claudin-b also exhibits greater transcript abundance in isolated goldfish PVCs than MRCs, supporting the notion of TJ heterogeneity between different gill cell types, even in FW fishes (Chasiotis et al., 2012). This latter observation takes on greater significance when considered together with the well accepted paradigm of gill TJ heterogeneity in SW (or SW-acclimated) fishes. Evidence from in vivo and in vitro studies has suggested the existence of claudins that are not associated with gill PVCs, but instead may only be associated with gill mitochondrion-rich cells and/or accessory cells. For example, puffer fish claudin-10d and -10e mRNA and Atlantic salmon claudin-10e transcripts were found to be significantly increased in the gills of SW versus FW-acclimated fishes (Bui et al., 2010; Tipsmark et al., 2008b). Consistent with a functional role for orthologous claudin-10 in mammalian models (Günzel et al., 2009), these observations suggest that claudin-10d and -10e are likely pore-forming proteins, and may partly account for the ‘leaky’ phenotype ascribed to TJs between adjacent MRCs and ACs in the SW (or SW-acclimated) teleost fish gill (see Section 4). In fact, while claudin-10d and -10e transcripts are detected in whole gill tissue from puffer fish, these salinity-responsive claudins are absent from cultured puffer fish PVC epithelia, and are currently presumed to be exclusively associated with gill ionocytes such as MRCs and/or ACs (Bui et al., 2010; Bui and Kelly, 2011). Claudin-6 has also been reported to be absent in cultured gill PVCs, but exhibits lower transcript abundance in SW versus FW fish gills (Bui et al., 2010; Bui and Kelly, 2011). This has led to the contention that claudin-6 may also be associated with gill ionocytes (in puffer fish at least) and that reduced abundance may promote cation movement through the MRC-AC ‘leaky’ paracellular pathway. This line of reasoning is supported by studies in MDCK cells, where transfection with claudin-6 resulted in elevated transepithelial resistance and a decreased sodium permeability-to-chloride permeability ratio (see Sas et al., 2008).

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275

Table 2 Claudins in the gill tissue and gill epithelial cells of teleost fishes and factors that are currently known to regulate their mRNA and/or protein abundance. Claudin

Species

Gill expression

Regulation

Refs.

Claudin-1

Takifugu rubripes Tetraodon nigroviridis Danio rerio (previously claudin-19)

Whole tissue Whole tissue, PVCs Whole tissue

Not known Not known Not known

Claudin-2 Claudin-3

Danio rerio Dicentrarchus labrax Oreochromis mossambicus Paralichthys lethostigma Fundulus heteroclitus Tetraodon nigroviridis

Whole tissue Whole tissue Whole tissue (claudin 3-like) Whole tissue (claudin 3-like) Whole tissue Whole tissue

pH Salinity Salinity Salinity Salinity Salinity, External Ca2+

Tetraodon biocellatus Oncorhynchus mykiss

PVCs Whole tissue Whole tissue PVCs

Cortisol Salinity Not known Cortisol, Dex

Danio rerio (=claudin-h)

Whole tissue

pH

Carassius auratus (=claudin-h)

Whole tissue, MRCs

Salinity

PVCs

Cortisol, fish serum

Takifugu rubripes Tetraodon nigroviridis

Whole tissue Whole tissue

Not known Salinity

Tetraodon biocellatus Danio rerio (=claudin-c) Carassius auratus (=claudin-c)

PVCs Whole tissue Whole tissue Whole tissue, MRCs

Not known Salinity pH Not known

PVCs

Cortisol

Oreochromis mossambicus Paralichthys lethostigma Fundulus heteroclitus

Whole tissue (claudin 4-like) Whole tissue (claudin 4-like) Whole tissue

Salinity Salinity Salinity

Loh et al. (2004) Bui and Kelly (2011) Kumai et al. (2011) and Clelland and Kelly (2010) Kumai et al. (2011) Boutet et al. (2006) Tipsmark et al. (2008a) Tipsmark et al. (2008c) Whitehead et al. (2011) Bui and Kelly (2011), Bagherie-Lachidan et al. (2008) and Pinto et al. (2010) Bui and Kelly (2011) and Bui et al. (2010) Duffy et al. (2011) Chasiotis and Kelly (2011a) Chasiotis and Kelly (2011a) and Kelly and Chasiotis (2011) Kumai et al. (2011) and Clelland and Kelly (2010) Chasiotis and Kelly (2012) and Chasiotis et al. (2012) Chasiotis and Kelly (2011a) and Chasiotis et al. (2012) Loh et al. (2004) Bui and Kelly (2011) and Bagherie-Lachidan et al. (2008) Bui and Kelly (2011) and Bui et al. (2010) Duffy et al. (2011) Kumai et al. (2011) Chasiotis and Kelly (2012) and Chasiotis et al. (2012) Chasiotis and Kelly (2011a) and Chasiotis et al. (2012) Tipsmark et al. (2008a) Tipsmark et al. (2008c) Whitehead et al. (2011)

Takifugu rubripes Tetraodon nigroviridis Takifugu rubripes Tetraodon nigroviridis

Whole tissue Whole tissue, PVCs Whole tissue Whole tissue, PVCs

Not known Not known Not known Not known

Loh et al. (2004) Bui and Kelly (2011) Loh et al. (2004) Bui and Kelly (2011)

Takifugu rubripes Tetraodon nigroviridis

Whole tissue Whole tissue Absent from PVCs Whole tissue

Not known Salinity

Loh et al. (2004) Bui and Kelly (2011) and Bui et al. (2010) Bui and Kelly (2011) and Bui et al. (2010) Kumai et al. (2011)

Takifugu rubripes Tetraodon nigroviridis Takifugu rubripes Oncorhynchus mykiss (=claudin-7)

Whole tissue Whole tissue, PVCs Whole tissue Whole tissue PVCs

Not known Not known Not known Not known Cortisol, Dex, RU486

Danio rerio (=claudin-7) Carassius auratus (=claudin-7)

Whole tissue Whole tissue

pH Salinity

PVCs, MRCs

Not known

Claudin-3a

Claudin-3c

Claudin-3d

Claudin-4

Claudin-5 Claudin-5a Claudin-5b Claudin-6

Danio rerio (=claudin-j) Claudin-7 Claudin-7a Claudin-7b

Claudin-8 Claudin-8a Claudin-8b Claudin-8c Claudin-8d

pH

Whole tissue

Cortisol

Danio rerio Takifugu rubripes Tetraodon nigroviridis Takifugu rubripes Tetraodon nigroviridis Takifugu rubripes Tetraodon nigroviridis Takifugu rubripes Tetraodon nigroviridis

Whole tissue Whole tissue Whole tissue, PVCs Whole tissue Whole tissue, PVCs Whole tissue Whole tissue, PVCs Whole tissue Whole tissue

pH Not known Not known Not known Not known Not known Not known Not known Salinity, external Ca2+

Oncorhynchus mykiss

PVCs Whole tissue PVCs

Not known Not known Cortisol, Dex, RU486

Loh et al. (2004) Bui and Kelly (2011) Loh et al. (2004) Chasiotis and Kelly (2011a) Chasiotis and Kelly (2011a) and Kelly and Chasiotis (2011) Kumai et al. (2011) Chasiotis and Kelly (2012) and Chasiotis et al. (2012) Chasiotis and Kelly (2011a) and Chasiotis et al. (2012) Chasiotis and Kelly (2012) Kumai et al. (2011) Loh et al. (2004) Bui and Kelly (2011) Loh et al. (2004) Bui and Kelly (2011) Loh et al. (2004) Bui and Kelly (2011) Loh et al. (2004) Bui and Kelly (2011), Bui et al. (2010), Pinto et al. (2010) and Bagherie-Lachidan et al. (2009) Bui and Kelly (2011) and Bui et al. (2010) Chasiotis and Kelly (2011a) Chasiotis and Kelly (2011a) and Kelly and Chasiotis (2011)

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Table 2 (Continued) Claudin

Species

Gill expression

Regulation

Refs.

Carassius auratus

Whole tissue, PVCs

Salinity

Whole tissue PVCs

Cortisol Fish serum

MRCs

Not known

Chasiotis and Kelly (2011a), Chasiotis and Kelly (2012) and Chasiotis et al. (2012) Chasiotis and Kelly (2012) Chasiotis and Kelly (2011a) and Chasiotis et al. (2012) Chasiotis et al. (2012)

Whole tissue Whole tissue Absent from PVCs Whole tissue Absent from PVCs Whole tissue

Not known Salinity

Claudin-10 Claudin-10c Claudin-10d

Takifugu rubripes Tetraodon nigroviridis

Claudin-10e

Tetraodon nigroviridis Salmo salar

Salinity Salinity, Cortisol, Smoltification

Claudin-11 Claudin-11a

Tetraodon nigroviridis

Claudin-11b

Danio rerio (=claudin-11)

Claudin-12

Claudin-13 Claudin-14 Claudin-14b

Loh et al. (2004) Bui and Kelly (2011) and Bui et al. (2010) Bui and Kelly (2011) and Bui et al. (2010) Bui and Kelly (2011) and Bui et al. (2010) Bui and Kelly (2011) and Bui et al. (2010) Tipsmark et al. (2008b) and Tipsmark et al. (2009) Tipsmark et al. (2008b)

Whole tissue PVCs Whole tissue

Salinity Cortisol Not known

Bui and Kelly (2011) and Bui et al. (2010) Bui and Kelly (2011) and Bui et al. (2010) Kumai et al. (2011) and Clelland and Kelly (2010)

Takifugu rubripes Tetraodon nigroviridis Oncorhynchus mykiss

Whole tissue Whole tissue, PVCs Whole tissue PVCs

Not known Not known Not known Cortisol, Dex, RU486

Danio rerio

Whole tissue

pH

Carassius auratus

Whole tissue

Salinity

PVCs

Cortisol

Takifugu rubripes Tetraodon nigroviridis

MRCs Whole tissue Whole tissue, PVCs

Not known Not known Not known

Loh et al. (2004) Bui and Kelly (2011) Chasiotis and Kelly (2011a) Chasiotis and Kelly (2011a) and Kelly and Chasiotis (2011) Kumai et al. (2011) and Clelland and Kelly (2010) Chasiotis and Kelly (2011a), Chasiotis and Kelly (2012) and Chasiotis et al. (2012) Chasiotis and Kelly (2011a) and Chasiotis et al. (2012) Chasiotis et al. (2012) Loh et al. (2004) Bui and Kelly (2011)

Takifugu rubripes

Whole tissue

Not known

Loh et al. (2004)

Takifugu rubripes Tetraodon nigroviridis

Whole tissue Whole tissue, PVCs

Not known Not known

Loh et al. (2004) Bui and Kelly (2011)

Claudin-23b

Takifugu rubripes Tetraodon nigroviridis Tetraodon nigroviridis

Whole tissue Whole tissue, PVCs Whole tissue PVCs

Not known Not known Salinity Not known

Loh et al. (2004) Bui and Kelly (2011) Bui and Kelly (2011) and Bui et al. (2010) Bui and Kelly (2011) and Bui et al. (2010)

Claudin-26

Takifugu rubripes

Whole tissue

Not known

Loh et al. (2004)

Takifugu rubripes Tetraodon nigroviridis

Whole tissue Whole tissue

Not known Salinity

Tetraodon biocellatus Salmo salar

PVCs Whole tissue Whole tissue

Cortisol Salinity Salinity, cortisol

Claudin-27b

Takifugu rubripes Tetraodon nigroviridis Tetraodon biocellatus Danio rerio (=claudin-f)

Whole tissue Whole tissue, PVCs Whole tissue Whole tissue

Not known Not known Salinity pH

Claudin-27c

Tetraodon nigroviridis

Whole tissue

Salinity

Claudin-27d

Anguilla Anguilla Takifugu rubripes Tetraodon nigroviridis

PVCs Whole tissue Whole tissue Whole tissue, PVCs

Cortisol Salinity Not known Not known

Loh et al. (2004) Bui and Kelly (2011) and Bagherie-Lachidan et al. (2009) Bui and Kelly (2011) and Bui et al. (2010) Duffy et al. (2011) Tipsmark et al. (2008b) and Tipsmark et al. (2009) Loh et al. (2004) Bui and Kelly (2011) Duffy et al. (2011) Kumai et al. (2011) and Clelland and Kelly (2010) Bui and Kelly (2011) and Bagherie-Lachidan et al. (2009) Bui and Kelly (2011) and Bui et al. (2010) Kalujnaia et al. (2007) Loh et al. (2004) Bui and Kelly (2011)

Takifugu rubripes Tetraodon nigroviridis Salmo salar

Whole tissue Whole tissue, PVCs Whole tissue

Not known Not known Prolactin

Oreochromis mossambicus Takifugu rubripes

Whole tissue Whole tissue

Salinity Not known

Claudin-19 Claudin-23 Claudin-23a

Claudin-27 Claudin-27a

Claudin-28 Claudin-28a

Claudin-28b

Loh et al. (2004) Bui and Kelly (2011) Tipsmark et al. (2008b) and Tipsmark et al. (2009) Tipsmark et al. (2008a) Loh et al. (2004)

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277

Table 2 (Continued) Claudin

Claudin-28c Claudin-29 Claudin-29a

Species

Gill expression

Regulation

Refs.

Tetraodon nigroviridis Oncorhynchus mykiss

Whole tissue, PVCs Whole tissue

Not known Not known

PVCs

Cortisol, RU486

Salmo salar

Whole tissue

Not known

Danio rerio (=claudin-e)

Whole tissue

pH

Carassius auratus (=claudin-e)

Whole tissue, PVCs, MRCs

Salinity

Whole tissue, PVCs

Cortisol

PVCs

Fish serum

Takifugu rubripes Tetraodon nigroviridis

Whole tissue Whole tissue, PVCs

Not known Not known

Bui and Kelly (2011) Chasiotis and Kelly (2011a) an Sandbichler et al. (2011a,b) Chasiotis and Kelly (2011a), Kelly and Chasiotis (2011) and Sandbichler et al. (2011a,b) Tipsmark et al. (2008b) and Tipsmark et al. (2009) Kumai et al. (2011) and Clelland and Kelly (2010) Chasiotis and Kelly (2011a), Chasiotis and Kelly (2012) and Chasiotis et al. (2012) Chasiotis and Kelly (2011a), Chasiotis and Kelly (2012) and Chasiotis et al. (2012) Chasiotis and Kelly (2011a) and Chasiotis et al. (2012) Loh et al. (2004) Bui and Kelly (2011)

Danio rerio (=claudin-d) Carassius auratus (=claudin-d)

Whole tissue Whole tissue

pH Salinity

PVCs

Fish serum

MRCs Whole tissue

Not known Not known

Kumai et al. (2011) Chasiotis and Kelly (2011a), Chasiotis and Kelly (2012) and Chasiotis et al. (2012) Chasiotis and Kelly (2011a) and Chasiotis et al. (2012) Chasiotis et al. (2012) Loh et al. (2004)

Claudin-29b

Takifugu rubripes

Claudin-30

Oncorhynchus mykiss

Whole tissue PVCs

Not known Cortisol, RU486

Salmo salar

Whole tissue

Salinity, Cortisol, RU486

Oreochromis mossambicus Takifugu rubripes Tetraodon nigroviridis Takifugu rubripes Tetraodon nigroviridis Takifugu rubripes Tetraodon nigroviridis Takifugu rubripes Danio rerio (=claudin-b)

Whole tissue Whole tissue Whole tissue, PVCs Whole tissue Whole tissue, PVCs Whole tissue Whole tissue, PVCs Whole tissue Whole tissue

Salinity Not known Not known Not known Not known Not known Not known Not known pH

Carassius auratus (=claudin-b)

Whole tissue, PVCs, MRCs

Salinity

Takifugu rubripes Tetraodon nigroviridis Oncorhynchus mykiss

Whole tissue Whole tissue, PVCs Whole tissue PVCs Whole tissue

Not known Not known Not known Cortisol, Dex, RU486 pH

Danio rerio (=claudin-i)

Whole tissue PVCs Whole tissue

Salinity Cortisol pH

Takifugu rubripes

Whole tissue

Not known

Bui and Kelly (2011) and Bui et al. (2010) Bui and Kelly (2011) and Bui et al. (2010) Kumai et al. (2011) and Clelland and Kelly (2010) Loh et al. (2004)

Takifugu rubripes Tetraodon nigroviridis

Whole tissue Whole tissue PVCs

Not known Salinity Cortisol

Loh et al. (2004) Bui and Kelly (2011) and Bui et al. (2010) Bui and Kelly (2011) and Bui et al. (2010)

Claudin-30a Claudin-30b Claudin-30c Claudin-30d

Claudin-31

Danio rerio (=claudin-g) Claudin-32 Claudin-32a

Claudin-32b Claudin-33 Claudin-33b

Tetraodon nigroviridis

Chasiotis and Kelly (2011a) Chasiotis and Kelly (2011a) and Kelly and Chasiotis (2011) Tipsmark et al. (2008b), Tipsmark et al. (2009) and Engelund et al. (2012) Tipsmark et al. (2008a) Loh et al. (2004) Bui and Kelly (2011) Loh et al. (2004) Bui and Kelly (2011) Loh et al. (2004) Bui and Kelly (2011) Loh et al. (2004) Kumai et al. (2011) and Clelland and Kelly (2010) Chasiotis and Kelly (2011a), Chasiotis and Kelly (2012) and Chasiotis et al. (2012) Loh et al. (2004) Bui and Kelly (2011) Kelly and Chasiotis (2011) Kelly and Chasiotis (2011) Kumai et al. (2011) and Clelland and Kelly (2010)

PVCs, pavement cells; MRCs, mitochondrion-rich cells; Dex, Dexamethasone.

Further studies, both in vivo and in vitro (see Section 7 for in vitro studies) also indicate and support a role for osmoregulatory hormones in the endocrine regulation of claudins within the teleost gill. For example, cortisol is an important osmoregulatory hormone in fishes that has been linked to the maintenance of salt and water balance in SW, FW and IPW (Laurent and Perry, 1990; McCormick and Bradshaw, 2006; Perry et al., 1992). In FW fishes, elevated cortisol levels have been reported to cause changes in gill epithelium morphology and transcellular transport properties that enhance ion uptake (Laurent and Perry, 1990; Perry and Wood, 1985; Perry

et al., 1992). This undoubtedly reflects a role for corticosteroids in the acquisition of ions in a FW environment. But it has also been shown that cortisol can reduce paracellular permeability and ion efflux rates across cultured FW fish gill epithelia (Kelly and Wood, 2001, 2002; Wood et al., 2002). In vivo, a cortisol-induced reduction in gill permeability may contribute to ionoregulatory homeostasis by limiting branchial ion loss and cortisol injections significantly elevated claudin-27a and -30 mRNA in the FW Atlantic salmon gill, but had no effect on these same claudins within the gills of SW-acclimated fish (Tipsmark et al., 2009). Similarly, when

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Table 3 ZO-1 expression in the gill tissue and gill epithelial cells of teleost fishes and factors that are currently known to regulate ZO-1 abundance. Species

Gill expression

Regulation

Refs.

Takifugu rubripes Tetraodon nigroviridis Carassius auratus

Whole tissue, pillar cells PVCs Whole tissue, PVCs, MRCs PVCs Whole tissue PVCs Whole tissue

Not known Not known Salinity Cortisol, fish serum Not known Dex, RU486 Not known

Kato et al. (2007) Bui et al. (2010) Chasiotis and Kelly (2011a, 2012) and Chasiotis et al. (2012) Chasiotis and Kelly (2011a) and Chasiotis et al. (2012) Kelly and Chasiotis (2011) Kelly and Chasiotis (2011) and Walker et al. (2007) Engelund et al. (2012)

Oncorhynchus mykiss Salmo salar

PVCs, pavement cells; MRCs, mitochondrion-rich cells; Dex, Dexamethasone.

intraperitoneal implants were used to elevate circulating cortisol levels in the FW goldfish, claudin-e, -7 and -8d mRNA abundance was increased in the gills (Chasiotis and Kelly, 2012). A comprehensive overview of claudins in the teleost gill tissue and gill cells is provided in Table 2. 5.3. ZO-1 in the gill epithelium of fishes ZO-1 was the first TJ protein discovered (Stevenson et al., 1986) and is arguably one of the most versatile TJ components identified. Frequently described as a TJ ‘scaffolding’ or ‘adaptor’ protein, ZO-1 is found on the inner cytosolic surface of TJs and has several binding domains which target other TJ proteins (e.g. occludin and claudins), cytosolic signaling molecules (e.g. transcription factors) and cytoskeletal actin (reviewed by Bauer et al., 2010) (see Fig. 3). It is believed therefore, that ZO-1 plays a dual role: (1) to provide indispensible structural support to TJs by linking occludin and claudins to the actin cytoskeleton; and (2) to transduce signals relating to gene expression, cell proliferation and differentiation via intracellular signaling pathways (Bauer et al., 2010). Currently, little is known about the functional role of ZO-1 in the teleost fish gill (see Table 3), however in vitro studies using primary cultured gill models have linked alterations in ZO1 mRNA abundance with reductions in paracellular permeability (see Section 7), a role which is supported by reports utilizing cultured mammalian epithelial models (e.g. Van Itallie et al., 2009). Given the more or less ubiquitous expression of ZO-1 transcript among goldfish tissues (Chasiotis and Kelly, 2012), it is likely that ZO-1 also supports the structure and organization of TJs in all teleost epithelia. ZO-1 has been localized to the periphery of gill pavement cells in rainbow trout (Walker et al., 2007) and spotted green puffer fish (Bui et al., 2010). In the gills of FWacclimated Atlantic salmon, ZO-1 immunostaining was co-localized with Na+ –K+ -ATPase and detected along gill filament and lamellar surfaces, therefore suggesting associations with both MRCs and PVCs (Engelund et al., 2012). A similar ZO-1 staining pattern was also observed in the SW-acclimated salmon gill, however little Na+ –K+ -ATPase co-localization was observed, which was proposed to indicate negligible ZO-1 associations with SW-type MRCs (Engelund et al., 2012). In the FW goldfish, ZO-1 transcript abundance was found to be significantly greater in isolated PVCs when compared to MRCs (Chasiotis et al., 2012). Also, both PVC and MRC fractions isolated from IPW-acclimated goldfish gills exhibited significantly greater ZO-1 transcript abundance when compared to fractions obtained from FW fish gills (Chasiotis et al., 2012). Despite this observation, ZO-1 mRNA abundance in whole goldfish gill tissue was significantly reduced in response to IPW acclimation, suggesting that ZO-1 alterations may have also occurred in other gill cells, e.g. cells of the gill vasculature (Chasiotis et al., 2012). In this regard, ZO-1 has been shown to immunolocalize to gill pillar cells in Fugu (Kato et al., 2007) and taken together, these observations highlight the importance of considering contributions from the gill vasculature when utilizing whole gill tissue for ZO-1 mRNA and protein abundance studies.

6. Surrogate gill models and paracellular permeability Directly measuring the permeability properties of the fish gill epithelium is experimentally challenging. This is partly due to the architectural complexity of the gill, which makes electrophysiological endpoints difficult to determine. In addition, cellular heterogeneity in the gill also creates problems. From a TJ standpoint, there are two facets to this latter variable. First is the cellular heterogeneity of the gill epithelium itself, which makes it difficult to pinpoint how ‘tight’ or ‘leaky’ TJs may be between different cell types. Second is the cellular heterogeneity of the gill as a tissue. In this regard, whole gill tissue possesses a prominent vasculature, the cells of which are linked by TJs. Issues of architectural complexity were overcome by the identification and use of surrogate gill models such as the opercular epithelium (Karnaky et al., 1977; Foskett and Scheffey, 1982; Foskett et al., 1981; Marshall, 1985), cleithral epithelium (Marshall et al., 1992) or jawskin epithelium (Marshall, 1977). Derived from the branchial/pharyngeal region of fishes, these architecturally simple epithelial sheets are largely composed of cells typically found in the gill. As surrogate gill models, they have been the foundation upon which major advances in our understanding of gill transepithelial ion transport have been based. Indeed, it is the electrophysiological data from these models that provides keystone information in support of the ‘leaky’ SW gill versus ‘tight’ FW gill paradigm (e.g. Foskett et al., 1981). Nevertheless, these ex vivo preparations still exhibit cellular heterogeneity and have a limited life span following isolation. Another approach is to use primary cultured gill epithelia that are ‘reconstructed’ on permeable polyethylene terephthalate membranes at the base of cell culture insert cups (for review, see Wood et al., 2002). These preparations are architecturally simple epithelia that exhibit paracellular permeability characteristics closely mimicking those of the intact gill epithelium and other surrogate gill models (see Kelly et al., 2000; Wood et al., 2002). Cell culture insert cups are housed in cell culture wells, and because the cultured epithelium separates an apical and basolateral compartment, endpoints of permeability such as transepithelial resistance (TER) or paracellular tracer flux can be easily measured. To overcome some of the issues associated with cellular heterogeneity, primary cultured gill epithelia can be ‘reconstructed’ using gill PVCs (Wood and Pärt, 1997) or gill PVCs and MRCs (Fletcher et al., 2000). Furthermore, cultured preparations do not contain cells derived from the gill vasculature. Taken together, these characteristics make them well suited for the study of TJs in the gill epithelium. To date, cultured gill preparations have been developed from diverse species including rainbow trout (O. mykiss) (Wood and Pärt, 1997), sea bass (Dicentrarchus labrax) (Avella and Ehrenfeld, 1997), tilapia (Oreochromis niloticus) (Kelly and Wood, 2002), goldfish (C. auratus) (Chasiotis et al., 2010) and spotted green puffer fish (T. nigroviridis) (Bui and Kelly, unpublished observations).

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7. Tight junction proteins in surrogate gill models Given that primary cultured gill epithelia allow alterations in TJ protein abundance to be assessed in association with measured changes in permeability, as well as in the absence of cells from the gill vasculature, these in vitro gill models can provide important mechanistic data regarding the function and regulation of TJ proteins in the teleost gill. For example, when siRNA were used to reduce occludin mRNA and protein abundance within a cultured goldfish PVC model, permeability to a paracellular marker was significantly increased (Chasiotis et al., 2012), thus supporting in vivo observations and the contention that at least one role for occludin in the gill epithelium of teleost fishes is to act as a barrier-forming TJ protein (see Chasiotis and Kelly, 2008; Chasiotis et al., 2009; Section 5.1). Cortisol has been demonstrated to significantly reduce paracellular permeability of cultured gill epithelia as well as passive ion ‘loss’ (efflux) across these preparations (Kelly and Wood, 2001, 2002; Wood et al., 2002). In recent studies, greater insight into the molecular physiology of this phenomenon has been uncovered. Cortisol treatment was shown to significantly increase TJ depth between cultured PVCs, elevate occludin mRNA and protein abundance as well as up-regulate transcript abundance of several claudins and ZO-1 (Chasiotis et al., 2010; Chasiotis and Kelly, 2011a; Kelly and Chasiotis, 2011; Sandbichler et al., 2011b). These studies have indicated a ‘tightening’ role for claudins previously uncharacterized in piscine models such as claudin-7, -12, and -31 as well as ZO-1, and support the in vivo characterization of occludin and claudin-3a, -8d, 28b, and -30 as barrier-forming proteins in the gill epithelium (see Sections 5.1 and 5.2). Corticosteroid receptor agonist treatment of cultured FW trout PVC epithelia also suggested that transcription of certain TJ proteins in the gill can be stimulated by cortisol through both glucocorticoid and mineralocorticoid receptor types (e.g. occludin, claudin-7, -8d, -12, -31 and ZO-1) or individual glucocorticorticoid (e.g. claudin-3a) or mineralocorticoid (e.g. claudin-28b, -30) receptors (Kelly and Chasiotis, 2011). Increased claudin-10e, -27a and -30 transcript abundance in response to cortisol treatment has also been reported for ex vivo Atlantic salmon gill preparations (i.e. gill explants) (Tipsmark et al., 2009). Considering the well-described role of cortisol in restructuring of the euryhaline fish gill for salt secretion in a hyper-osmotic setting or ion-uptake in a hypo-osmotic environment (reviewed by Evans et al., 2005), these studies collectively suggest that cortisol may additionally enhance the function of the gill during osmotic stress by ‘tightening’ TJs between PVCs at least, in order to limit passive salt gain or loss during SW or FW acclimation respectively. Indeed, in the cultured FW rainbow trout PVC model, cortisol treatment was found to additionally modulate the dynamic remodeling of claudin-28b protein localization and distribution that was observed to occur during osmotic stress and recovery (Sandbichler et al., 2011a). Furthermore, since corticosteroid-mediated alterations in TJ proteins have been documented in various mammalian epithelial and endothelial models (e.g. Förster et al., 2008; Stelwagen et al., 1999), the results of studies using primary cultured gill models indicate that certain elements of the endocrine regulation of TJs in vertebrates may be evolutionarily conserved. The specific response of gill TJ protein orthologs to cortisol however was not found to be the same in all species of fishes examined. Doses of cortisol equal to those that elicit robust changes in the cultured gill epithelium permeability of trout in the aforementioned studies elicited only a small ‘tightening’ effect in cultured goldfish PVC epithelia, with little to no alterations in occludin or orthologous claudin transcripts (Chasiotis and Kelly, 2011a). These results suggest that cortisol may play a limited role in the endocrine regulation of paracellular permeability across the stenohaline teleost gill, and also provide insight into

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species-specific differences in the endocrine control of TJs that may relate to the environmental physiology of teleost fishes (e.g. euryhaline versus stenohaline). It has been found that the paracellular permeability properties of cultured gill epithelia are responsive to supplementation with serum derived from fishes in different physiological states (Kelly and Wood, 2002; Chasiotis et al., 2012). This can provide in vitro insight into paracellular properties of the gill that would otherwise be difficult to ascertain. For example, alterations in cultured gill epithelium permeability brought about by cortisol or dexamethasone treatment can also be observed when serum derived from stressed fish is used to supplement culture media (Kelly and Wood, 2002). Furthermore, in recent studies a cultured goldfish PVC model was supplemented with serum derived from goldfish acclimated to FW or IPW (Chasiotis et al., 2012). Cultured gill preparations supplemented with IPW-serum exhibited increased TER and decreased flux to a paracellular marker, and demonstrated a greater paracellular barrier to apical IPW exposure than those treated with FW-serum (Chasiotis et al., 2012). These changes in permeability occurred in association with elevated occludin and claudin-e and -h transcript abundance (Chasiotis et al., 2012), therefore supporting their roles as barrier-forming TJ proteins and their involvement in gill restructuring under hypo-osmotic conditions.

8. Conclusion and perspectives The main purpose of this review is to consolidate information on TJs in the gills of fishes as well as research that seeks to understand the varying physiological and molecular properties of TJs in the gills of teleost fishes. In the case of the latter, this area is emerging as complex beyond expectation. Moving forward, initial challenges present themselves in the form of species specific TJ protein enumeration as well as screening TJ elements to evaluate which respond to alterations in environmental and/or systemic variables. For example, in teleosts the claudin family of TJ proteins has only been fully enumerated in the puffer fishes Fugu (=Takifugu) rubripes and T. nigroviridis, and in each of these species there are over 30 claudins in the gill tissue (see Table 2). It can be anticipated that large numbers of claudins will also be present in the gills of other species and in some cases this is already evident (e.g. D. rerio, see Table 2). In addition to the claudins, there are other transmembrane and cytosolic TJ proteins to be considered (e.g. occludin, ZO-1, -2, -3, cingulin etc.), most of which have yet to be described in any fish species. Once TJ proteins are identified in a species of interest, an assessment of their response to environmental and or/systemic change can isolate specific candidate proteins for further study. At this stage, the challenge of working with a complex tissue such as the gill epithelium will continue to be demanding in so far as all work with the architecturally complex, heterogeneous gill epithelium presents its obstacles. However, some of these issues can be overcome by the use of in vitro models and/or techniques that have already helped to make sense of the complexities of fish gills (see previous sections). In addition, there is no question that greater insight into the specific roles of TJ proteins in the gill epithelium of fishes will be enhanced by the adoption of ‘loss of function/gain of function’ techniques as well as the broader availability of ever more sophisticated experimental tools associated with the increasingly popular piscine model D. rerio. However, it is also important to remember that fishes represent almost 50% of all extant vertebrates with an evolutionary story that unfolds over approximately half a billion years. Therefore, as an incongruent assemblage containing both agnathans and gnathostomes, fishes are a diverse group that inhabit a very broad array of environments, As such, it will also be very exciting to see the gill TJ story develop in diverse teleost species as well as non-teleost species.

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Acknowledgements This work was supported by a NSERC Discovery Grant as well as a NSERC Discovery Accelerator Supplement, Canadian Foundation for Innovation New Opportunities Grant, and Ontario early researcher award to SPK. HC received Ontario Graduate Scholarship funding.

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