‘Fish matters’: the relevance of fish skin biology to investigative dermatology

Authors


Prof Dr Charli Kruse, Fraunhofer Research Institution for Marine Biotechnology, Paul-Ehrlich-Street 1-3, 23562 Lübeck, Germany, Tel.: 0049-451-38444810, Fax: 0049-451-38444812, e-mail: charli.kruse@emb.fraunhofer.de

Abstract

Please cite this paper as:‘Fish matters’: the relevance of fish skin biology to investigative dermatology. Experimental Dermatology 2010.

Abstract:  Fish skin is a multi-purpose tissue that serves numerous vital functions including chemical and physical protection, sensory activity, behavioural purposes or hormone metabolism. Further, it is an important first-line defense system against pathogens, as fish are continuously exposed to multiple microbial challenges in their aquatic habitat. Fish skin excels in highly developed antimicrobial features, many of which have been preserved throughout evolution, and infection defense principles employed by piscine skin are still operative in human skin. This review argues that it is both rewarding and important for investigative dermatologists to revive their interest in fish skin biology, as it provides insights into numerous fundamental issues that are of major relevance to mammalian skin. The basic molecular insights provided by zebrafish in vivo-genomics for genetic, regeneration and melanoma research, the complex antimicrobial defense systems of fish skin and the molecular controls of melanocyte stem cells are just some of the fascinating examples that illustrate the multiple potential uses of fish skin models in investigative dermatology. We synthesize the essentials of fish skin biology and highlight selected aspects that are of particular comparative interest to basic and clinically applied human skin research.

‘…the diverse inhabitants of our world are just variations on a theme.’

Neil Shubin: Your Inner Fish, 2009

Introduction: the piscine-mammalian divide in current skin research

Vertebrate skin with its outermost layer, the epidermis, separates the individual from its environment and is one of the organism’s crucial interfaces for contact and communication with its external milieu. As a veritable multi-purpose tissue, skin and its appendages serve numerous essential functions. These range from thermoregulation, sensory functions, hormone synthesis as well as metabolism and maintenance of fluid balance, osmotic homeostasis, via inter-individual communication by visual signals (pigments, hair), the elaboration of special structures (claws, nails, hair) and production of various specific substances (e.g. pheromones, antimicrobial peptides) to glandular secretions and the protection against a large variety of biological, chemical and physical stressors (1–3). This amazing portfolio of diverse functions has developed only gradually, as the vertebrate variations of skin that we know today have had uncounted precursors during evolution. The surviving species on this planet provide only a meek glimpse into the full spectrum of cutaneous phenotypes that must have been generated throughout evolution.

However, if asking most current-day dermatologists what they spontaneously associate with ‘fish’, a term that encompasses all aquatic, non-tetrapod vertebrates with or without scales, it is not ancestral precursors of our skin, but – besides ichthyoses – most likely the potential applications of fish oil preparations in future dermatological therapy (e.g. 4,5), and fish protein or fish parasite-associated allergic skin disorders (6,7). This illustrates the degree to which, over the last few decades, mammalian skin research has essentially lost not only contact with fish skin biology, but also with the leads and lessons that can be distilled from a careful re-evaluation of the piscine origins in general. Although there are a few notable exceptions (most prominently in the study of zebrafish melanoma 8,9), this development is both regrettable and ill-advised. Additionally, there are some interesting features of adult stem cells (SC) in fishes regarding the regeneration processes, which have been demonstrated, e.g. for fin-, heart- or eye regeneration. These processes, especially skin damages, should be further investigated and then compared to the human integument. In the suggestive wording of a recent book that explores our connections to the piscine universe, it is about time that we re-discover ‘our inner fish’ (10). Along this vein, the current review argues that it is both rewarding and important for investigative dermatology to revive its interest in fish skin biology. We list specific reasons why fish skin permits the study of a cornucopia of problems that are also of major relevance to mammalian skin, delineate specific examples for potential uses of fish skin models in investigative dermatology, and provide a – necessarily eclectic – synthesis of those aspects of fish skin biology that are of particular interest to basic and clinically applied human skin research with a comparative biology edge.

Why fish skin biology matters in investigative dermatology

Besides the attractions that come along with tracing-back the evolutionary origins of one’s favourite object of study (10) and the well-appreciated reasons for turning to the zebrafish in melanoma research (covered elsewhere: e.g. 8,9,11), there are multiple other reasons for turning one’s attention to the biology of fish skin:

  • • The last common ancestor of fish and mammals is probably more than 350 million year old if one considers lungfish, and more than 420 million years in the case of zebrafish. Despite this enormous temporal divide in evolution, fish skin shows architectural similarities with that of mammalians, even though it follows specific construction principles that are perfectly adapted to aquatic life: similar to the epidermis of terrestrial vertebrates such as mice or man, the fish epidermis is a multilayered tissue that is separated from the dermis by a distinct basement membrane (12,13). It is, however, generally a ‘mucous’ and not a ‘keratinized’ system (Figs 1–3). Studying fish skin in vivo (in aquaculture or laboratory fish tanks) can thus be more convenient and/or instructive for addressing selected basic questions relating to epidermal biology than in human subjects. To name just two examples, the increasing interest in water channel protein (aquaporin) functions in human epidermis could optimally be complemented by studying ancestral aquaporin functions in fish skin in vivo (14), just as tight junction research in mammalian epidermis could draw inspiration from recent insights into, e.g. claudin expression and function in fish skin (15).
  • • For cutaneous in vivo experimentation, the availability of spontaneous and engineered fish mutants with defined skin phenotypes, namely in the zebrafish, has made fish models an increasingly attractive alternative to mutant mice: Large scale, comparatively inexpensive mutagenesis and screening strategies have already generated zebrafish models of a wide variety of human diseases, including some skin disorders, and are now being increasingly employed in drug discovery and drug development programmes (16).
  • • It is an advantage of fish model-based skin research that here, the entire development of skin can be conveniently and comprehensively studied within a very short time window, e.g. by time-lapse imaging, from the larval stage to the adult fish (17). Useful markers for studying, e.g. zebrafish epidermal development, have also been defined (18,19) that facilitate comparison with mammalian epidermal development. Employing such markers, conserved mechanisms between fish and mammalian skin development can now conveniently be explored.
  • • The usefulness of piscine in vivo-genomics for mammalian skin research notably includes developmental skin biology, as the generation of vertebrate skin appendages (scales, feathers, hairs) shares a number of common developmental pathways, such as Hedgehog, BMP and Wnt signalling pathways, which have been conserved from the earliest beginnings to the current-day pinnacle of vertebrate evolution (20,21). Novel insights into the functions of Hedgehog signalling in fish (22) might serve as a discovery tool for investigative dermatology. Further, it has recently been uncovered that abnormalities in ectodysplasin/ectodysplasin receptor (EDA/EDAR) signalling that cause human hypohidrotic ectodermal dysplasias (3,21) and the rs-3 mutant phenotype in Medaka, which results in a disturbed development of most scales involve the same TNF pathway (23).
  • • Further similarities between piscine and mammalian developmental biology extend to the molecular controls of melanocyte SC and their progeny. Genes like wnt, Sox10, Mitf, Kit and Slf are now appreciated to drive the development of pigmentation patterns in zebrafish (24–26), while their mammalian homologues apparently control melanocyte stem cell functions, e.g. in mouse and human hair follicles (27). Thus, development and homeostasis of melanocytes via replenishment by a stem cell pool may be an evolutionarily conserved strategy in pigment pattern development (24,28), where researchers active in one system can only learn from those in the other.
  • • Mammalian and fish skin share crucial protective functions, namely against infection. In particular, fish skin offers a unique opportunity to study the origins of innate antimicrobial defense systems. As piscine skin expresses a large variety of antimicrobial peptides (AMPs) such as hepcidin (29), defensin-like peptides (30), certain apolipoproteins (31) and piscidin (32), often with selective properties against pathogenic bacteria, fungi, algae, viruses or parasites (33,34), the ever-increasing body of published work on fish AMPs deserves much greater attention by the research community interested in the innate immune system of mouse and human skin (35–37). Which of these fish skin AMPs are still utilized by human skin? Which new functions have they attained during evolution? What can ancestral functions of fish-derived AMPs, whose homologues are also found in human skin, teach us about the full range that specific AMPs may still exert in our epithelium? It is known from aquacultured fishes that a breakdown of the slime layer resulted in affection by micro-organisms and fungi, which can be compared with human infections.
  • • The immune system of teleost skin has many additional features that may be of interest for the study of infectious diseases affecting human skin. For example, the skin of some fish has elaborated intracutaneous antibody-secreting B lymphocyte-like cells that are an integral component of the fish immune system and that confer long-term, parasite antigen-specific humoral immunity against reinfection by the same parasite (38). Besides the emerging evidence for similarities between fish skin immune responses to parasite infection (39) and the immune defense of human skin against various parasites (40), are there lessons to be learned on how to enhance the efficacy of intracutaneous humoral immunity in the human system where plasma cells are a prominent clinical feature (e.g. in syphilis and borreliosis)?
  • • The recent renaissance of cutaneous (neuro-) endocrinology in the mammalian system (e.g. (41) could be further promoted by a reappraisal of fish skin (neuro-)endocrinology within investigative dermatology. To list just one example, members of the pituitary growth hormone family of peptide hormones [growth hormone, prolactin, somatolactin (Table 1)] operate as crucial modulators of osmoregulation, growth, metabolism and possibly chromatophore function in fish skin (42–44). This should offer new indications to the as yet quite incompletely explored functions of the homologues of these peptide hormones that are generated, e.g. in human skin (cf. 45).
  • • Even stress research in the mammalian system, which investigates the impact of perceived stress on skin (46), is likely to be aided by systematic cross-referencing to piscine stress models. In fact, just as in mammals, stress responses in fish consist of the sympathetic nervous system activation and increased release of CRH (47–49). Fish responding to the stressful conditions of aquaculture or transport have increased serum cortisol levels and, interestingly, even up-regulate heat shock protein expression in their skin (50).
  • • As some human epithelia continuously face a wet environment, there are obvious similarities to fish skin: Dermatologists frequently deal with infectious diseases of oral mucosal surfaces comprising salivary proteins. Therefore, it may well be of dermatological interest that mucus IgM is an important player in the mucus-associated immune system of fish skin, e.g. in fugu, which employs the same basic immunoglobulin transport system as mammalian intestine (146). Perhaps, the anti-infection potential of fish mucus could be exploited in topical dermatological therapy, in and beyond infectious diseases of the oral mucosa?
  • • Finally, fish have an extraordinary regenerative capacity that dwarfs anything seen in the mammalian system, especially in humans. Zebrafish, for example, show an impressive array of tissues that can be regenerated including fins, spinal cord, optic nerve, heart and skin (51). Mammalian skin wound healing research may profit from the increasing molecular insights distilled from, e.g. the regrowth of amputated caudal fins (52), where comprehensive gene expression data bases are becoming available (53). Given the central role of angiogenesis in mammalian skin regeneration, this, too, could be complemented by zebrafish models for the study of blood vessel formation (54). Still, a good deal more can be learned about the underlying molecular basis of fish skin regeneration (55–57), and this could be recovered and harnessed in healing-impaired human skin.
Figure 1.

 Schematic drawings of structural principles of skin. (a) the skin of the teleost fishes with dermal scales (Original drawing by M. Emde (according to 156), (b) human skin. 1:sweat gland, 2:sebaceous gland, 3: arrector pili muscle, 4: blood vessel.

Figure 2.

 Tissue sections of rainbow trout and cod skin stained with Aldehydfuchsin-Goldner. (A) Young rainbow trout (Oncorhynchus mykiss, approximately 8 weeks old). Section showing the epidermis (1), dermis (2), hypodermis (3) and part of the musculature (M). Strata: stratum superficiale (a), stratum spinosum (b), stratum basale (c), stratum laxum (d), stratum compactum (e). Scale bar: 300 μm. Arrowhead indicates mucus glands, which release mucus to the epidermal surface. (B) Cod (Gadus morhua). Section of dorsal skin showing the epidermis (1) and dermis (2). BC:basal epithelial cells, BV: blood vessel, EC: superficial epithelial cells, FB: fibroblast-like cells, MG: Mucus glands. Scale bar: 500 μm. * spaces are arteficial because of cutting procedure. (C) Adult rainbow trout (Oncorhynchus mykiss). 1 – dermis, 2 – scales, 3 – pigment cells, 4 – subcutis, 5 – dense fibrous tissue, stratum compactum, HE-staining, scale bar 1200 μm. Epidermal parts are not shown.

Figure 3.

 Skin of adult cod (Gadus morhua). E – epidermis, ScP – scale pocket. PAMS (periodic acid methenamin silver)-staining. Asterisk – large goblet cell.

Table 1.    (Neuro-)endocrinology of fish epidermis: Hormones that are well-known in teleost fish skin. Most of the (partial neuroendocrinically) released hormones, like inhibitory or stimulating hormones, thyroid hormones, prolactin and isotocin (Vasopressin-Oxytocin-family) are produced by the adenohypophysis
HormoneProduced byClassificationKey function(s) in fish skinReferences
Melanin-concentrating hormone (MCH)Pituitary glandPeptide hormoneLightens skin colour by stimulating aggregation of melanosomes(125)
Melanocyte-stimulating hormone (MSH)Pituitary glandPeptide hormoneAntagonistic effect to MCH, causes dispersion of melanosomes(126)
SerotoninClub cells and Merkel cellsPeptide hormoneInvolved in pathogen defence, stimulates exocrine glands to secrete amine messengers(118)
ProlactinPituitary glandPeptide hormoneAffects thickening of epidermis, increases number of goblet cells, important role in osmoregulation, intensifies coloration of skin(44,47,127,128)
Thyroid hormones (e.g. thy-1)Thyroid glandThyroid hormonesEssential for the embryonic and postembryonic development, important hormones for metamorphosis in flatfish and also other finfish(129,130)
GonadotropinsPituitary glandPeptide hormoneDecrease number of goblet cells in marine teleosts(131)
CorticosteroidsAdrenal cortexSteroid hormoneCortisol influences hydromineral balance, energy metabolism and immune function, corticosterol and growth hormone have stimulatory effects on epithelial cell secretion but do not affect goblet cells(132,133)
Melatonin and noradrenalineGonads, testesSteroid hormoneIncrease the skin transparency, effect decoloration in combination with prolactin, show positive effects on both, transparency and coloration(44)
TestosteroneGonads, testesSteroid hormoneIncreases epidermal thickness, 11-ketotestosterone decreases number of superficial goblet cells(134–136)
EstrogensGonads, testesSteroid hormoneNo direct effects on skin known, but high concentrations of oestrogens or derivatives in water may be routed through the skin(137)
Epinephrine and norepinephrineAdrenal glandsCatecholaminesEpinephrine more common and may control chloride cells in fish by lowering cAMP-levels(63)
Substance pSensory cellsNeuropeptideNeuromasts react in some teleosts, as well as some ampullary organs (electroreceptors) and taste buds(138)
Thyrotropin-releasing hormone (TRH)Pituitary glandNeuropeptideTRH stimulates growth hormone, and prolactin release, stimulator of alpha-MSH release(139)

These selected arguments should suffice as encouragement to reappraise the usefulness and promise of fish skin models for investigative dermatology. Let us turn, then, to the salient features of fish skin biology (for detailed reviews, see e.g. 58–62).

The emergence of fish: a major evolutionary achievement

Fishes are subdivided into three large recent groups: Agnatha (recent only the Cyclostomata), Chondrichthyes (with the Holocephala and the more important Elasmobranchii, i.e. sharks and rays) and Osteichthyes. The latter are again subdivided into the small group of the Sarcopterygii (ancestors of Tetrapoda, with the lungfish) and the very large group of the Actinopterygii (with the most important subgroup, the Teleostei). The Teleostei with their more than 30 000 species are the most numerous group of all vertebrates (Fig. 4). One major achievement of fishes is certainly the formation of the neural crest, found only in chordates. From this channel-like structure the neural tube is established, which is situated between epidermis and notochord. Cells of the neural crest migrate to different parts of the embryo, where they differentiate into diverse types of cells, like parts of the teeth, cartilage or sensory cells of the skin. Likewise, several different types of pigment cells arise from the neural crest, including yellow xanthophores, reflective iridophores and black melanocytes (24). This evolutionary achievement is linked with important features of fish skin: a stratified mucogenic epidermis and an alpha-keratogenic potential.

Figure 4.

 Possible tree of the evolution of vertebrates. Non-vertebrates in blue, teleosts in red, e.g. rainbow trout and cod, and examples of evolutionary achievements in yellowish boxes. Asterisks adjacent to name of taxon indicate these are known only as fossils.

In striking contrast to mammals, a cornified cellular envelope is restricted to specific body regions (e.g. barbel) or found only in some teleosts such as, e.g. Periophthalmus or the Syngnathidae (sea horses), which have some ‘cornified’ adaptations, because the epidermis of fish mostly does not contain proteins connected with interkeratin matrices and corneous cell envelope formations (filaggrin, loricrin). These intermediate filaments are the basis for stabilization of mammalian epidermal cells forming a largely water-impermeable apical layer. Instead, fish epidermis is covered by a layer of mucus, a slimy layer of glycoproteins that is heavily enriched with antimicrobial factors, including antibodies, complement, lysozyme, C-reactive protein, lectins, proteases, transferrin and polypeptide antibiotics (63).

Construction principles

The skin of all fish species, like that of any other vertebrate, consists of two basic layers: an outer, the epidermis, and an inner, the dermis (Figs 1 and 2). The entire outer surface of a fish, including the body and fins, is completely covered by epithelium. Beneath its protective mucus, which is also very important against hydrodynamic drag, cell-cell-junctions of the stratified squamous superficial epithelial cells, also called filament cells or Malpighian cells, provide the only epithelial coherence. Investigating the functions of tight junctions in fish skin could be directly relevant for investigative dermatologists, because the role of tight junctions, which are expressed not only by keratinocytes but also by epidermal Langerhans cells, is of outstanding recent interest in human epidermal biology (2,150,151). In fish, this boundary is interrupted by mucus-secreting cells. All of these cells are part of the stratum superficiale, the outermost part of three epidermal strata, followed by the stratum spinosum medial with differentiated cells and proximal the stratum basale with basal cells and the basement membrane. The thickness of these epidermal structures is often bound to ecological factors, to seasonal variances or to variances between males and females. Undifferentiated epidermal progenitor cells emerging from the basal layer are induced to proliferate and differentiate in the stratum spinosum when needed and subsequently recruited to the outermost epidermal layer. Depending upon the fish species, fish age, location on the body, epidermis thickness and number of epidermal layers, various specialized cells, including goblet cells, sensory cells, alarm cells and chloride cells may be present in the epidermis (61).

Fish epidermis is separated from the underlying dermis by a layer of filamentous proteins, which form the basement membrane.

The adjacent dermis is composed of the stratum laxum and the stratum compactum and, in striking contrast to mammalian skin, is separated from the hypodermal adipogenic tissue by yet another endothelial layer, called the dermal endothelium (64). The thin stratum laxum of fish dermis consists of a loosely arranged connective tissue, complemented with blood vessels and nerve fibres. Dermal cells are mostly fibroblasts, interspersed with different chromatophores. Scale-building cells, the scleroblasts of fish skin, are arranged in the scale pockets. The most frequent scale type in teleosts is the elasmoid scale (Fig. 3), which consists of a plate of collagenous tissue, with superficial mineralization, surrounded by scleroblasts and fibroblasts (61). On the lower side, the scale pocket is lined by modified fibrocytes with desmosomes and caveolae. Bundles of collagen fibres anchor the scale in its pocket. The posterior edge of the scale is more or less covered by the epidermis, depending on the species and their biology.

Regions of dermo-epidermal interactions in the fish skin may be present like it is supposed for reptilians (148). Here, morphoregulatory molecules are exchanged and may then have significant influence on the structural composition of the epidermis and dermis. In mammals, small dermo-epidermal connected regions migrate into the dermis and form dermal papillae and then hairs (148).

Fish epidermis: from stratification via mucus secretion to anti-infection defense

The composition of the three aforementioned epidermal strata is invariably the same in all teleost fishes. The stratum superficiale acts as a seal between the animal and its surroundings. Its surface is ornamented with species-specific microridges (62), which are raised, actin-rich structures that serve to maintain the mucus layer on the surface of the fish (60). The cells of the stratum superficiale are stabilized by microfilaments like 70A α-keratin. While this type of keratin is abundantly found in all fish epitheliocytes, ß-keratin is absent. In almost all fish species, the epidermis does not have a dead, keratinized surface as in terrestrial vertebrates, but consists entirely of living cells. An inverse correlation between mucus secretion and keratinization is suggested (65).

As in mammals, fish skin is metabolically very active. Physiological functions of fish skin include temperature regulation – cold-water fish like the antarctic cod, possess epithelial cells with specific antifreeze proteins (66) – in some cases nutrient uptake, or the repair of surface wounds. Immediately following an injury, cells from the margins close the wound by secreting mucus, which transports lymphocytes into the damaged area (55). Evidently, it would be interesting to compare the underlying mechanisms with human wound healing so as to define basic, common principles. A covering epithelial layer forms much more rapidly in fishes than in warm-blooded animals but after the wound is covered further recovery in fishes is extremely slow (67).

The mucus is produced by goblet cells of the stratum spinosum. Their structure is similar to that of mammalian goblet cells (68). Mucus goblet cells die when they release their glycoconjugates, hence there is a continuos turn over in the outer layers of the epidermis. Alongside the mucus goblet cells, club cells and sensory cells are embedded in this layer. These cells exhibit enormous metabolic reaction capacities to meet various external influences (65).

Fish skin possesses a fascinating multitude of other specialized cells (see also Table 2) with various functions, just to mention the lateral line system to measure distances or to provide active electro location for orientation in the dark (69,70). Somatosensory receptors on the head of rainbow trout can be classified based on their responses to touch, pressure, heat and chemical stimulation (71). They include polymodal nociceptors with properties similar to those found in mammals (71–73). It is tempting to speculate that a systematic neurophysiological study of fish skin nociceptors could reveal new insights into the neurobiological mechanisms that underlies clinical problems such as ‘sensitive skin’ (152), neuropathic itch (153) and complex regional pain syndrome (CRPS) (154). Chemical substances that are dissolved in water can be detected by chemosensory systems, which allow the recognition of mates, conspecifics, predator or prey; they mediate migration and homing and support reproductive and feeding activities. The products of club cells are not secreted; these cells store proteins and acidophilic substances. They are often species-specific and are also known as ‘Schreckstoffzellen’, taking account of the experimentally supported hypothesis that when the skin is damaged by a predator, these alarm pheromones are released into the water and warn conspecifics of the predator’s presence (74,145).

Table 2.   Cell types in teleost fish skin detected by histological methods
Cell typeLocationDefinitionKey function(s)Present in atlantic cod (Gadus morhua)Present in rainbow trout (Onco-rhynchus mykiss)Present in zebrafish (Danio rerio)Equivalent in human skinReferences
  1. +, detected/present; -, not detected/present; ?, doubtful/unknown.

Mucous goblet cellEpidermisSerous glandSecretion, protection+++Cornified envelopes(61)
Club cell (alarm cell)EpidermisSecretory cellAlarm system, pheromonal function?-/?-/?+-(61,118)
Epithelial cellEpidermisEpitheliumStability, gas exchange, protein source+++Keratinocytes(62,65,81)
Basal (epithelial) cellEpidermisEpithelium, undifferentiated progenitor cellsAttachment to dermis, drive differentiation+++Basal epithelial cells(61)
Chemosensory cellsEpidermisSensory cellsSense of water current, dissolved chemical substances???- 
Merkel cellsEpidermisNeuroendocrine cellsHormonal function???Merkel cells(119)
FibroblastDermisConnective tissueMaintain the structural integrity of connective tissues, structural framework, wound healing+++Fibroblast(120,121)
MelanophoreDermisChromatophoreProtection against UV-radiation, coloration, colour changes, infection defense+++Melanophores(44,122)
IridophoreDermisChromatophoreLight reflection+++-(122)
XanthophoreDermisChromatophoreColoration-/?-/?-/?-(122)
Blood vesselDermisBlood capillaries (endothelial cells)Blood transport, supply+++Blood vessel(61)
NervesDermisAfferent nervesInnervation-/?-/?-/?Nerves(61)
Scales (elasmoid scales)DermisMineralized plates dervied from mesodermProtection, decrease of water flow resistance, calcium source, mechanical properties+++Hair, teeth(121,123,124)
Fat cell (adipocyte)Hypodermis (Subcutis)Mesenchymal cell (derived from fibroblasts?)Storage, isolation, movability of the skin relative to the musculature+++Fat cells(61)

However, most epithelial cells in the stratum spinosum remain non-differentiated, this is why this layer may be seen as a stem cell reservoir (see Box). Epithelial cells are not continuously peeled off, like outer epidermal cells of mammals, but only replaced by cells from the stratum spinosum upon death or injury. Thus, although the fish epidermis does not appear to be constantly renewed, homeostatic mechanisms must be in place to ensure maintenance of this tissue (60).

Antimicrobial defenses of fish skin

Fish live in an aquatic environment that is substantially richer in pathogens than the aerial environment of mammals. Hence, epidermal integrity is vital for defense, because ubiquitous pathogens can quickly colonize an open wound as they are present even on healthy skin, albeit in small numbers. For this reason, neutral and acid (carboxylated and sulphated) glycoconjugates are continuously secreted by fish skin as defence against invading pathogens (Figs 2A and 5). The composition of the carbohydrates changes with stress and environmental conditions (58,65,75,76). Additionally, free sugars and their antimicrobial abilities are used on the epidermal surface, whereby first information on such approach has been achieved from humans (155). The mucosal immunity is well described by Dickerson 2009 (143).

Figure 5.

 Schematic illustration of the antimicrobial defense mechanism in fish. When chemical or mechanical injury occurs, e.g. through bacteria, viruses or other pathogens, epithelial cells start to release cytokines like IL-1, which is suggested to attract phagocytes as well as T cells and B cells to the superficial parts of the epidermis. At the same time, mucus goblet cells start to secrete their products: mucus, complement factors, peptides, carbohydrates, immunoglobulins, lysozymes and proteases, carbohydrates and lectins. The immunoglobulins activate the complement pathway that leads to opsonization of the pathogens. Antimicrobial peptides, like IL 1β or Hepcidin, are involved in the immune response to bacteria, whereas neuropeptides, like Corticosterol or serotonin, affect the release of mucus. Ig: Immunoglobulins, IL-1: Interleukin 1, MHC: major histocompatibility complex. Black bar on the right represents thickness of the epidermis.

The mucus contains various further substances, as for example lysozyme (77), immunoglobulins (78), complement 10 (79), carbonic anhydrase (80), lectins (81), crinotoxins (82), calmodulin (83), C-reactive protein (84) and antibacterial peptides. Many AMPs that inhibit microbial growth by assembling pore forming units in the bacterial membrane have been discovered (85–89). These AMPs are highly conserved components of the innate immune system, with an ever-expanding repertoire (37,141,142). Further exploration of AMPs in fish skin is likely to reveal new candidate AMPs in the search for mammalian homologues and may provide new fish-derived AMPs that could become exploitable for human skin therapy, e.g. in patients with atopic dermatitis whose skin displays pathologically heavy colonization by bacteria. Lysozyme that functions by degrading the bacterial cell wall was found in rainbow trout and was shown to participate in the first-line defense of fish (90). Hepcidin, which plays an important role as a blocker of iron transport by inhibiting ferroprotein, has been recently sequenced by Shike et al. (91) in zebrafish. They found a remarkable conservation of aminoacid sequences and gene organization between zebrafish and other species. Interestingly, also trypsin was discovered in the mucus cells of atlantic cod (92). Many of these substances might be isolated and used for fish and human health-related applications (93,144).

Immunobiology of fish skin

As a second defensive barrier, an effective cellular immune system similar to that of humans seems to be a prerequisite for the fish’s survival. Both innate and adaptive immune responses are mounted by fish to control parasite infections, and several mechanisms described for mammalian parasitoses have also been demonstrated in teleosts (39). For example, epidermal migration of inflammatory cells and their secretions may counteract the establishment and proliferation of ectoparasites (94). Again, this invites one to search for basic lesions on principles of antiparasitic defence that may still apply to human skin.

Once the micro-organisms circumvent the non-specific immune defense of the mucous layer, the cell-mediated immunity of the body seems to take over, and there is a rush of phagocytes that engulf the pathogens and present them to the macrophages and neutrophiles. These in turn produce an inflammatory response via various cytokines and leukotriens leading to the accumulation of T cells that directly interact with the macrophages and are quite important for inducing both, antiviral defense and antibody production by B lymphocytes. T-cell activation is provided by costimulatory signals of CD80 and CD86 interacting with CD28 in mammals, while rainbow trout and other teleost fish contain a single CD80/86 gene, with highest degree of sequence conservation and phylogenetic relationship with CD80 and CD86 molecules (95). Rainbow trout is able to produce a localized mucosal immune response via systematically stimulated B cells that migrate to mucosal tissues where they produce antibodies (96). The equivalents of natural killer cells in fish are the non-specific cytotoxic cells (97) that are known to mediate innate immune responses. Immunoglobulins present in the circulation also activate the complement pathway that leads to opsonization of pathogens (Fig. 5).

Mast cells in fish are mostly present in the connective tissue of the intestine and skin. Their response to infection is similar to that of mammalian mast cells. Two studies of parasitized rainbow trout have shown that in long-term inflammatory reaction of intestine and skin, there is mast cell accumulation (98,99).

Why fish skin matters economically and technologically

Under the current, difficult conditions for obtaining adequate funding for skin research, it surely deserves mentioning that the funding potential for studies into fish skin is of substantial, yet under-appreciated interest to investigative dermatology. This mainly is driven by increasing awareness of the value of fish skin for different applications in medicine and industry. For the fish industry, especially the Teleostei are of outstanding economical importance. The worldwide total amount of fisheries in 2007 was about 140 000 000 t [95 mio. t capture and 45 mio. t aquaculture, (112)], making fish a major economical factor with total imports estimated at 100 billion $ for 2008 [from the Globefish database, http://www.globefish.org/, FAO Fisheries and Aquaculture Department (eds), accessed 20 March 2009].

By 2020 more than a third of the yearly fish production will come from aquaculture farms. Because of the restricted space in aquaculture facilities, very high stocking densities of fish cause an increased susceptibility to pathogens. Therefore, understanding the fish immune defense via its skin is of enormous importance. In contrast, the economic use of fish skin is currently only important for the production of fishoil and fishmeal, which explains why biotechnical applications have long been scarce.

However, recognition of the vital importance of healthy skin for fish survival and thriving, not the least in aquaculture, is changing this inferior economic image of fish skin. This is further changed by emerging industrial and biomedical applications of fish skin. An example for this is the isolation of antibacterial and antifungal compounds found in high concentrations, e.g. in the mucus or epidermal extracts of pollack, flounder and sole (113). They were found to be active against gram-negative and gram-positive bacteria and in parts against fungi (113). Conceivable uses in clinical dermatology might include skin disorders located in intertriginous skin with their often massive overcolonization with various skin bacteria and Candida albicans. Further examples are fish skin-derived gelatine, e.g. from grass carp (114), and the multiple medical and cosmetic applications of fish collagen as a new biomaterial substitute for living tissues (115), which may replace bovine collagen once the haemostatic properties, immunogenicity and cytocompatibility of fish skin collagen have been satisfactorily evaluated.

Novel biomedical applications (e.g. cytotoxicity testing or transgenic zebrafish to understand, e.g. various protein pathways) will also arise from fish cell cultures, which are simple and safe to handle and react fast, e.g. to chemical exposure (116). Taken together, these examples suggest that the systematic exploration of fish skin models, is not only biologically, clinically and technologically relevant, but will also open interesting new funding opportunities for investigative dermatology.

Box: the enigma of fish skin stem cells

Stem cells are important in the development and homeostasis of a variety of vertebrate tissues. Recently, different tissue niches have been identified as sources for adult SC (100–102) including human skin (103,104), and skin stem cell biology is a rapidly advancing field in the biotechnology and life science sector. Skin represents a large reservoir for adult SC, including mesenchymal, hematopoietic (105) and neural SC (106). Because of its permanent renewal and high propensity to repair, the epidermis is a very good tissue to explore stem cell biology. Despite the relative simple construction of fish epidermis, its SC are not as well understood as in humans, where the complexity of the epidermis showed the heterogeneity of the stem cell compartment. Further, mammalian skin cell lines are well established (107–109), but no fish skin cell line has been developed so far. However, fish cells in long-term culture showed characteristics of stem/progenitor cells [(147); for a good overview on stem cells in cartilaginous and bony fish, see (140)].

The rare, slowly cycling, multipotent ‘bulge’ SC in the hair follicles and the more restricted interfollicular, follicle-matrix and sebaceous-gland SC generate a large pool of transit-amplifying progeny in higher vertebrates (110). These adult stem cells are restricted to a defined tissue compartment, the stem cell niche. Niches of epithelial stem cells in fish have been proposed to exist in that part of the epithelium from which a new tooth germ buds off (149), but might occur in, e.g. scale pockets as well.

The ability for constant self-renewal in fish epidermis rests on the presence of epidermal SC in the basal layer. Furthermore, the epidermis is regularly remodelled, retaining a strict balance between proliferation and differentiation. To participate in tissue regeneration and repair, SC need to be multipotent and, in the case of fish epidermal SC, be able to differentiate into epithelial cells, mucus glands, pigment cells or sensory cells. Like other SC, they are supposed to be slow-cycling in vivo, can self-renew, and are responsible for the long-term maintenance of the epidermis. Activated by wounding, they start to proliferate and to regenerate the tissue (51). Once established in an in vitro culture (111), adult SC of fish skin could be differentiated into tissues of interest and those cells used for different applications, such as in test systems for cytotoxic substances, on scaffolds or, if upscaling is possible and cost effective, as a substitute for fish meal (Fig. 6). To induce differentiation of SC, the identification of extra- and intracellular factors that influence this differentiation process is of crucial interest.

Figure 6.

 Diagram showing the isolation and proliferation of fish adult SC in vitro and their possible applications. (a) Adult donor fish. (b) Primary culture. (c) Adherent Cell culture. (d) Transfection with a transgene construct. (e) Selected progenitor cells, which could be used for test systems, scaffold growth and pellets or to produce adult chimeric fish, e.g. by microinjection (f). Diagram modified after Alvarez et al. (117).

Given that fish skin is immunologically privileged because of its high content of mucus proteins, fish skin SC will not only provide hope for the functional repair of the skin itself, but cell cultures will offer a potential source for cell-based therapies of injuries and diseases throughout the body of both fish and mammals, and maybe 1 day fish skin stem cell cultures will allow the isolation of potential therapeutic compounds and their large-scale production.

Acknowledgements

We are particularly grateful to Anna Emilia Petschnik for her comments and to Hermann Neumann from the Department of Marine Science, Senckenberg Institute, as well as to the Research Institute for Agriculture and Fisheries Mecklenburg-Vorpommern in Born/Darß for providing the fish skin material. Samples of fish skin for Cod (Fig. 2B) were obtained on the research cruise SO592 of the ‘Solea’, Johann Heinrich von Thünen-Institute. Writing of this review was made possible in part by grants from the European Fond for Regional Development (EFRE) to Charli Kruse and from Manchester NIHRS Biomedical Research Center to Ralf Paus.

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