From frog integument to human skin: dermatological perspectives from frog skin biology.

Biol. Rev. (2014), 89, pp. 618–655. doi: 10.1111/brv.12072

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From frog integument to human skin: dermatological perspectives from frog skin biology Iain S. Haslam1,∗ , Eric W. Roubos2 , Maria Luisa Mangoni3 , Katsutoshi Yoshizato4,5 , Hubert Vaudry6,7 , Jennifer E. Kloepper8 , David M. Pattwell9 , Paul F. A. Maderson10 and Ralf Paus1,8 1

The Dermatology Centre, Salford Royal NHS Foundation Trust, Institute of Inflammation and Repair, University of Manchester, Oxford Road, Manchester, M13 9PT, U.K. 2 Department of Anatomy, Radboud University Medical Centre, Geert Grooteplein Noord 2, 6525 EZ, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands 3 Department of Biochemical Sciences, Istituto Pasteur-Fondazione Cenci Bolognetti, La Sapienza University of Rome, Piazzale Aldo Moro, 5-00185, Rome, Italy 4 Academic Advisors Office, Synthetic Biology Research Center, Osaka City University Graduate School of Medicine, Osaka, Japan 5 Phoenixbio Co. Ltd, 3-4-1, Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan 6 European Institute for Peptide Research, University of Rouen, Mont-Saint-Aignan, Place Emile Blondel 76821, France 7 INSERM U-982, CNRS, University of Rouen, Mont-Saint-Aignan, Place Emile Blondel 76821, France 8 Klinik für Dermatologie, Allergologie und Venerologie, Universitätsklinikum Schleswig-Holstein, Ratzeburger Allee 160, 23538 Lübeck, Germany 9 Leahurst Campus, Institute of Learning & Teaching, School of Veterinary Science, University of Liverpool, Neston CH64 7TE, U.K. 10 Department of Biology, Brooklyn College of CUNY, Brooklyn, NY 11210, U.S.A.

ABSTRACT For over a century, frogs have been studied across various scientific fields, including physiology, embryology, neuroscience, (neuro)endocrinology, ecology, genetics, behavioural science, evolution, drug development, and conservation biology. In some cases, frog skin has proven very successful as a research model, for example aiding in the study of ion transport through tight epithelia, where it has served as a model for the vertebrate distal renal tubule and mammalian epithelia. However, it has rarely been considered in comparative studies involving human skin. Yet, despite certain notable adaptations that have enabled frogs to survive in both aquatic and terrestrial environments, frog skin has many features in common with human skin. Here we present a comprehensive overview of frog (and toad) skin ontogeny, anatomy, cytology, neuroendocrinology and immunology, with special attention to its unique adaptations as well as to its similarities with the mammalian integument, including human skin. We hope to provide a valuable reference point and a source of inspiration for both amphibian investigators and mammalian researchers studying the structural and functional properties of the largest organ of the vertebrate body. Key words: frogs, skin structure and function, secretion, barrier, antimicrobial peptides, neuroendocrinology, wound healing, dermatology, osmoregulation, pigmentation. CONTENTS I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aims and structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Frog skin: environmental sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional anatomy and cytology of frog skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Ontogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Author for correspondence (Tel: +44 (0) 161 3060515; E-mail: [email protected]). Biological Reviews 89 (2014) 618–655 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society

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Frog skin biology

V.

VI.

VII. VIII. IX.

X. XI.

XII. XIII. XIV. XV.

619

(a) Frogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (b) Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Epidermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Epidermal mucus production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4) Dermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (5) Hypodermis (subcutis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (6) Innervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (a) Lateral line system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (b) Merkel cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (c) Thermoreceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (d) Rohon-Beard neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (e) Frontal organ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (f ) Vascular system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (g) Exocrine gland innervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristic features and novel adaptations of frog skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Minimising water loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Cocoon formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Water uptake by aquaporins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4) Gas exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (5) Predator repellents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (6) Chemical defence alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (Neuro-)endocrinology of frog skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Thyroid hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Thyrotropin-releasing hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Prolactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4) Cholecystokinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (5) Insulin-releasing factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (6) Pheromones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Further secretory activities of frog skin: AMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Frog skin immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Mast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Frog skin pigmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Chromatophore cell types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) α-MSH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Melatonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4) Comparison with mammalian chromatophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tools for frog skin research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Translational aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Barrier function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Anti-infection defence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4) Aquaporins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (5) Sebaceous and mucus glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (6) Tissue regeneration/wound healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supporting information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION Research into the biology of frogs is currently very extensive, encompassing numerous fields, such as cell biology, embryology, neuroscience, (neuro)endocrinology, ecology, genetics, behavioural science, evolution, public health, and conservation biology (e.g. Lutz, Blodt & Kloas, 2005;

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Vaudry et al., 2005; Hayes et al., 2006; Roubos et al., 2010a). Frogs belong to the order Anura, which also includes toads. Members of the family Ranidae, such as the European common frog (Rana temporaria), bullfrog (R. catesbeiana or Lithobates catesbeianus), green frog (R. clamitans), leopard frogs (R. pipiens), marsh frogs (R. ridibunda), pickerel frog (R. palustris) and wood frog (R. sylvatica), are generally considered ‘true’ frogs

Biological Reviews 89 (2014) 618–655 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society

620 because of their bulging eyes, long hind legs, large webbed hind feet, smooth or slimy skin, and egg-laying in clusters. On the other hand, ‘true’ toads like Bufo marinus and Bufo bufo belong to the Bufonidae and are identified by short hind legs, wart-like poison glands behind the eyes, a characteristic chest cartilage, a dry, leathery skin with bumps that break up the animal’s outline to blend visually into its environment, and egg-laying in long chains. However, for certain species such features are inconspicuous, or mixed so that they fall into both categories. A well-known example of the latter is Xenopus laevis, which is sometimes referred to as the South-African clawed toad, at other times as the South-African clawed frog. In this frog skin review, we will not make a distinction and include data about ‘mixed’ frogs and ‘true’ and ‘mixed’ toads. There are many reasons for the wide scientific interest in frogs. Their study can yield fundamental knowledge about primary physiological processes such as organ development and tissue regeneration, neuron functioning, epithelial cell growth and differentiation, metabolism, and reproduction. Furthermore, many frog species are endangered, which requires in-depth ecological research in order to understand the adverse environmental agents that threaten them. Thus, frogs can also serve as biomarkers for adverse environmental conditions (pollutants, toxicants, overall water quality/oxygenation). This can be of relevance to other vertebrates; for example, certain hormones found in frogs are conserved in humans and their study has strong implications for both conservation and human health. Indeed, by studying the effects of hormone analogues and other chemicals on frog development and behaviour, we can enhance our ability to evaluate environmental contamination. In this review we will focus on one particular aspect of frog biology that is of interest for human research and health, namely the structure and diverse functions of frog skin. It is likely that frogs and mammals shared a common tetrapod ancestor in the late Devonian, approximately 400 million years ago (mya). Figure 1 places frog and human evolutionary relationships in context and the evolution of amphibian skin is discussed in online Appendix S1. Among frog species, skin form varies considerably. Gross examination of frog skin reveals diverse phenotypes, from very smooth, multi-coloured surfaces to integuments rich in spiky or even hair-like protrusions (Fig. 2). A comparison of amphibian skin to our own reveals notable differences. So, why should anyone with a primary interest in human skin structure and function care about this peculiar frog organ, which evolved nearly 250 mya (Carroll, 2009)? An answer to this question lies in the existence of easily recognisable similarities between the skin of frogs and that of mammals, including man, in terms of anatomical features, physiological processes and molecular homologies (as discussed in detail below). The usefulness of frog research in life sciences is exemplified by progress in the field of anuran genomics (xenomics; Amaya, 2005). Whereas the mitochondrial genome of the most widely studied frog Xenopus laevis, has been available for some time (Roe et al., 1985), more

Iain S. Haslam and others recently the whole genome of its smaller family member X. tropicalis (Fig. 2F) has been reported (Hellsten et al., 2010), allowing detailed comparisons of gene nucleotide sequences and (putative) protein expression patterns not only between frog species but also between frogs and mammals. Major progress is also being made with elucidating frog peptidomics and proteomics, permitting frog-mammalian comparisons of peptide and protein expressions within specific cell types (e.g. Conlon et al., 2007a; Devreese et al., 2010; Marteil et al., 2012; Mechkarska et al., 2012). In addition, many new lines of transgenic and gene knockout frogs (e.g. Loeber, Pan & Pieler, 2009; Robert, Goyos & Nedelkovska, 2009; Scheenen et al., 2009; Love et al., 2011; Ishibashi, Cliffe & Amaya, 2012) and novel genetic engineering strategies such as remobilisation of ‘sleeping beauty’ transposons (Yergeau et al., 2011) provide powerful new tools for obtaining insights into the general principles of vertebrate cell differentiation and signalling. A recent Xenopus white paper gives an excellent overview of both historical and recent frog studies in functional genomics and genetics, relevant to biomedical research (Khokha, 2012), and another article in the same special issue of Genesis provides a useful guide to Xenopus genomics specifically (Abu-Daya, Khokha & Zimmerman, 2012). Beyond the level of the genome, numerous key principles that apply to mammalian skin biology can also be investigated in frog skin. For example, frog skin allows one to study the general principles of epidermal development, remodelling, defence and shedding (Alibardi, 2006; Lillywhite, 2006). As such, a systematic look at the frog epidermis may greatly facilitate attempts to understand the structure and function of basic skin elements (junctional and cytoskeletal proteins) that are so closely linked to the development and organisation of mammalian skin architecture (Kirschner et al., 2013; Simpson, Patel & Green, 2011; Yoshida et al., 2013). Another example can be found in the invention of a device owing a significant debt to frog skin, namely the ‘Ussing chamber’ (Ussing & Zerahn, 1951). This seminal experimental system laid the basis for a wide range of applications in the basic research of vectorial ion transport through frog skin, the integrity of epithelial barriers and the invasive properties of cancer cells. These applications were made possible as a result of the ease with which frog skin’s membrane resistance, current, voltage, impedance and capacitance can be measured with this equipment (e.g. Smith, 1975; Harvey, 1992; Hillyard, Cantiello & van Driessche, 1997; Nagel, Somieski & Katz, 2002). For instance, using the Ussing chamber, isotonic secretions from frog mucus glands (Ussing, Lind & Larsen, 1996) are much easier to study than human exocrine and apocrine secretions and, therefore, may yield important new insights into the functioning of mammalian sweat glands. Whilst frog skin provides an excellent model system with which to perform comparative studies with mammalian skin, it is important to note that recent advances in the technology and utility of human skin equivalents (i.e. Auxenfans et al., 2009; Felder, Goyal & Attinger, 2012) have resulted in


Frog skin biology

621

apodans c.180 spp.

BIRDS

Siderops 187 mya

REPTILES

fossil taxa

MAMMALS

FROGS & TOADS c. 6000 spp.

newts & salamanders c.600 spp

Latimeria

Hylonomus 312 mya Neoceratodus

LISSAMPHIBIA

Kotlassia 251mya basal amniotes

Triadobatrachus 247 mya

Eusthenopteron 380 mya

(C) fossil taxa

Panderichthys 382 mya

Capetus 310 mya

Temnospondyls

Coelacanth fish

Anthracosaurs

(B) Tiktalik 378 mya Lungfish

AMPHIBIA Polish marine trackways 395 mya

Choanate fish

Tetrapodamorphs

KERATINOCYTE LEGEND A, B & C plasma membrane

Guiya 419 mya

nucleus

increasing density α-keratin filaments Actinopterygii “ray-finned”

Sarcopterygii “lobe-finned”

mucous granules external mucous

(A) lipogenic lamellar granule

Osteichthyes

Finned BONY FISH

4-limbed TETRAPOD

corneous envelope external lipid deposit

Fig. 1. Legend on the next page.


622 physiologically relevant models of the mammalian epidermis and oral mucosa (Liu et al., 2010). These equivalents may now survive long-term culture (Stark et al., 2006; Boehnke et al., 2007) and have been adopted by commercial organisations for assessing, for example, transepidermal drug delivery (Pappinen et al., 2012), cytotoxicity and inflammation, and lend themselves well to high-throughput screening of potential therapeutic drugs (Lebonvallet et al., 2010). Nevertheless these human in vitro systems cannot accurately mimic the great complexity of the skin in vivo (such as the presence of dermal appendages). Consequently, intact animal models (in vivo as well as ex vivo) still hold a marked advantage over three-dimensional skin-equivalent cultures (Meier et al., 2013). In this respect, frogs do offer some unique advantages over other model species. A pertinent example is the extensive research into the biochemistry of frog skin secretions in relation to antimicrobial peptides (AMPs) and other bioactive molecules (Zasloff, 2002; Mangoni, Marcellini & Simmaco, 2007; Brogden & Brogden, 2011). The wide range of functions exerted by some AMPs (beyond their antimicrobial activities) ranges from antiviral (HIV) to antitumor (antiproliferative, angiostatic) to wound-healing-promoting

Iain S. Haslam and others actions (Bradbury, 2005; Raghavan et al., 2010; van Zoggel et al., 2010). This makes frog skin a useful model to screen for novel AMP functions evolutionarily preserved in human skin. From a practical point of view it is worth noting that AMPs can be readily isolated in large quantities from frog skin without the need to sacrifice the animal. Yet frog skin is not only a source of AMPs; it also provides a unique repository of other powerful bioactive molecules that, speaking in terms of evolution, continue to operate in mammalian skin. For instance, neuropeptides (e.g. the tachykinins) identified in mammals are also present in frog skin (Kastin, 2013) and some were even first discovered in frog skin (e.g. bombesin; Erspamer, Erspamer & Inselvini, 1970; Wharton et al., 1978) or detected using a frog skindarkening assay (α-melanophore-stimulating hormone; αMSH; Hogben & Winton, 1922). Moreover, in terms of evolution, neuroendocrine control of human epidermal mitochondrial function and biogenesis, i.e., by thyrotropin (TSH) (Poeggeler et al., 2010) and by thyrotropin-releasing hormone (TRH) (Knuever et al., 2012), a tripeptide abundant in frog epidermis (Jackson & Reichlin, 1977, 1979; Giraud et al., 1979) appears to date back at least to frogs (Galas et al., 2009). Indeed, inspired by the frog skin

Fig. 1. Homo sapiens considers frogs: an evolutionary context for frogs and toads (anuran lissamphibians) and man with comments on basic epidermal cytology. Black font is used for taxa and/or lineages with living members; red font for those represented only by fossils. For some fossils age is given as million years ago (mya, black font). None of the species, photographed or reconstructed, are represented to scale. Numbers of species for the three lissamphibian orders are approximations taken from www.amphibiaweb.org. This simplified phylogeny of vertebrates shows: osteichthyan and tetrapod lineages to the left and right, respectively, of the green dotted line; relationship between the Lissamphibia (top centre) and Homo sapiens (top right quadrant); three different forms (A, B, C) of epidermal keratinocyte occurring across the osteichthyan/tetrapod lineages: a key is shown in the legend box (bottom right). The oldest-known bony fish Guiya (Zhu et al., 2009), a lobe-fin sarcopterygian was a cousin of the earliest ray-finned fish, and an ancestor of the living Coelacanth (Latimeria), the Australian lungfish (Neoceratodus), and tetrapods. Choanate fish, known only as fossils, showed great diversity of body form within a short geological period: all had scaled integuments. It is inferred that species of all these ‘piscine’ lineages had a simple, stratified epidermis wherein epithelial cells were primarily mucogenic (A), the mucous being extruded from cells at the body surface. The solid red arrow indicates the age (395 mya) of a relative of the last common ancestor of the Amphibia and the amniote classes (mammals, reptiles and birds) known from trackways in marine flat sediments (Niedzwiedzki et al., 2010). It is inferred that basal amphibians, temnospondyls (centre) and anthracosaurs (centre right) had a multistratified epidermis wherein some degree of α-keratinization occurred and intercellular mucous facilitated cell-cell adhesion (B). Constituent cells had a corneous envelope but lacked the keratin-associated proteins and lamellar granules that characterise epidermal cells in living amniotes. Lissamphibians evolved from a temnospondyl ancestor (C). Two hundred and fifty mya: inter-relationships among the three major groups are subject to ongoing debate (Sigurdsen & Green, 2011). Epidermal structure seen in living species is built around the cell type illustrated in B. The life history of most frogs and salamanders involves an aquatic larval phase (indicated by cartoons) and the transition to the sub-adult involves metamorphosis. Reconstructions of anthracosaurs suggest a body form resembling that of modern lizards: all showed skeletal adaptations implying an active, terrestrial lifestyle and this is supported by analyses of fossil environments. From the grooves that lateral line sensory systems leave on skull bones, it is inferred that all fossil amphibian taxa had aquatic larvae and probably underwent metamorphosis during their development. Hylonomus can be identified as the first true amniote by its skeletal features and lack of lateral line scars on its skull bones: it is inferred that its reproduction involved the amniote egg (see pre-hatching turtle embryo, top right) seen in all living amniotes, albeit modified in mammalian viviparity. What cannot be determined is whether lipogenic lamellar granules (C), the basis for waterproofing skin in living amniotes, first appeared in bona fide amniotes such as Hylonomus, or whether they also characterised ‘advanced’ anthracosaurs such as Kotlassia. Further discussion of the data illustrated can be found as online Supporting Information (see online Appendix S1). The taxonomy of, and evolutionary inter-relationships within, tetrapodamorph groups named on this figure are subject to ongoing debate: the interested reader should consult Carroll (2009) and Clack (2012) for further details. Attribution of images in this figure in alphabetical order: Capetus (Bogdanov, 2007); Ceratophrys (Iberri, 2004); Dermophis (Andreone, 2006); Eusthenopteron (Tamura, 2007a); Guiya (Weasley, 2009); a herring (Chap, 2004); Hylonomus (Tamura, 2007b); Kotlassia (Bogdanov, 2000); Latimeria (Fernandez, 2007); Neoceratodus (Flower, 1898); Panderichthys (Tamura, 2007c); Siderops (Bogdanov, 2008); the Thinker (Lifson, 2010); Tiktalik (Tamura, 2007d); Triadobatrachus (Riha, 2007); Triton alpestris (Linder, 2005); turtle embryo hatching (Mayer, 2004). Biological Reviews 89 (2014) 618–655 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society

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(B)

(A)

(C)

(D)

(E)

(G)

(F)

Fig. 2. Diversity of body form and integumentary morphology in anuran lissamphibians. Within the order Anura, there is an enormous number of extant species showing a vast array of skin architecture with unique functional adaptations. As of 28 September 2013, www.amphibiaweb.org lists 6333 anuran species, constituting 88% of all known amphibians. (A) The Surinam horned frog Ceratophrys cornuta, one of 88 species of South American ceratophrynids, with numerous hyperkeratotic regions manifested as denticles, head horns, and ‘claws’ (arrows) (Jenkins, 2010). (B) The American common toad Bufo americana (Cephas, 2009): 1 of 569 bufonid species found world-wide in non-polar regions except Australia. In Australia the marine-tolerant anuran the cane toad B. marinus has been introduced: it has the dubious distinction of being a voracious, invasive pest whose skin secretions are lethal to many potential predators. (C) The Lake Titicaca frog Telmatobius culeus, 1 of 104 species of New World leptodactylid whose ‘typically frog-like’ skin is thrown into folds (arrows) that act as accessory respiratory devices (Oxford, 2006). (D, E) The hairy frog Trichobatrachus robustus, 1 of 144 species of sub-Saharan arthroleptids, has so-called ‘hairs’ on the lateral body and femoral regions that also act as accessory respiratory devices. Tissues in the rectangle in D are shown at higher magnification in E (Janzen, 2010; Dénes, 2011). (F, G) Two of 33 species of pipid frog native to Africa and South America but now found world-wide as medical research tools and invasives (Harland, 2010). Figure F is the diploid, tropical clawed frog Xenopus tropicalis and figure G is the much larger, tetraploid, South African clawed toad X. laevis. Biological Reviews 89 (2014) 618–655 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society

Iain S. Haslam and others

624 literature which proposed that intracutaneously generated TRH stimulates the pituitary production of melanotropic hormones that then act back on frog skin to regulate its pigmentation (Vaudry et al., 1999), the role of TRH in human skin has been investigated in more detail. This led to the discovery that TRH is a potent stimulator of mammalian melanogenesis and human hair follicle energy metabolism (Gaspar et al., 2011; Vidali et al., 2013). Moreover, by following such leads from frog skin literature, TRH has been identified as a potent promoter of human hair growth in vitro (Gaspar et al., 2010), a neuroendocrine stimulator of mitochondrial activity and mitochondrial biogenesis in human epidermis (Knuever et al., 2012), as a neuroendocrine regulator of human keratin expression (Ramot et al., 2013) and as a promoter of human skin re-epithelialisation (Meier et al., 2013). All of this progress in mammalian skin research has significantly contributed to bridging what may be described as the ‘amphibian-mammalian divide’ in skin research. The above considerations, plus others that will be discussed in detail later, amply illustrate why the fields of amphibian and mammalian skin biology as well as of investigative dermatology could all benefit greatly from systematic studies on frog skin.

II. AIMS AND STRUCTURE Since space restrictions preclude detailed coverage of skin structure and function across all living amphibians, we will consider integumentary features specifically shared by frogs and mammals. Therefore, this review guides the reader through the main concepts and current research frontiers of frog skin biology, which we hope will help to stimulate interest in comparative vertebrate skin biology (e.g. Maderson, 2004; Wu et al., 2004; Alibardi, 2006; Mikkola, 2007; Aspengren et al., 2009; Rakers et al., 2010; Rubin et al., 2010; Bongiovanni, Muller & Della Salda, 2011). While we recognise that some other amphibian species have also proven to be excellent experimental models, this review will focus only on frogs, as among amphibians they have been investigated in most detail. After briefly exploring the environmental sensitivities of frog skin, its developmental biology and basic construction principles, we will highlight comparative aspects of frog skin versus mammalian (and particularly human) skin properties. To do this, particular focus will be on skin ontogeny, neuroendocrinology, ion and water transport, exocrine secretion, immunology, wound healing and pigmentation. We then delineate important experimental tools and techniques available to mammalian skin researchers to study frog skin. Finally, we will explore key areas in which translational research, utilising frog skin models, could benefit human skin health. Additional online Supporting Information complements the core text by presenting information on aspects of frog skin evolution, development,

structure and function that are not covered in depth in the main body of this review (see online Appendices S1–S4).

III. FROG SKIN: ENVIRONMENTAL SENSITIVITY Biomonitoring, whereby environmental pollution is tracked by using living organisms as ‘sensors’, is increasingly applied as an insightful method for determining the impact of pollutants on particular ecosystems (e.g. Whitfield, 2001). In this field, frogs have become widely studied models (e.g. Falfushinska, Romanchuk & Stolyar, 2008) and their skin acts as a ‘biomarker’ for the overall levels of local pollution (Fenoglio et al., 2006). As the skin is the first organ to come into contact with environmental pollutants, it is not surprising that pronounced environmental effects on its structure and function have been described. Indeed, in a study of the frog, R. klepton esculenta, living in mildly versus heavily polluted rice fields, Fenoglio et al. (2006) identified skin differences in both ultrastructure (decreased keratinisation in the most polluted habitat) and in detoxifying enzyme levels. It is evident therefore, that such biomarkers can serve as a measure of both the degree of environmental pollution as well as the general viability of the anuran population (Venturino et al., 2003). Assessing the health of frog populations is increasingly important when one considers that amphibians form ‘the most threatened vertebrate class on the planet’ (Fisher, Garner & Walker, 2009). Although pollution and habitat destruction is an important threat to these animals, the primary cause of the widespread decline in the number of frog species is infection by Batrachochytrium dendrobatidis (Bd). This fungus colonises and thrives within frog skin epidermal cells ultimately resulting in death by chytridiomycosis. Indeed, although visible signs of the disease appear relatively mild (hyperkeratosis, increased epidermal shedding, infrequent skin lesions), the impact is extremely severe, postulated to result from the release of fungal toxins that interfere with cutaneous water homeostasis (Voyles et al., 2007; Fisher et al., 2009). As the maintenance of fluid and electrolyte balance is an important function of frog skin, disturbances to this process can have strong detrimental repercussions. This is highlighted by recent evidence that, upon Bd infection, Na+ transport is reduced, a key pathological feature of chytridiomycosis (for a recent review, see Campbell et al., 2012).

IV. FUNCTIONAL ANATOMY AND CYTOLOGY OF FROG SKIN (1) Ontogeny Before describing adult frog skin form and function in more detail, the developmental process of frog skin following fertilisation and throughout embryogenesis is described below, with particular reference to X. laevis. Subsequently,


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Fig. 3. Generation of deep cells in the developing frog embryo. Histology shows when deep cells are generated from the initially single-layered embryo. Serial sections were prepared of Xenopus laevis embryos at each division from the 32-cell embryo to the 1024-cell embryo (late stage 8), and stained with Ehrlich’s haematoxylin (Chalmers et al., 2003). In the 32-cell embryo, all blastomeres are superficial cells and contact both the external surface and the blastocoel. However, one cell division later, 64-cell embryos often have at least one deep cell (arrow). The number of deep cells increases through the next few cell divisions, and at the 512-cell stage more than one inner layer is present. At late stage 8, the inner layers are beginning to thin out to one layer by radial intercalation [see the inner layer around the vertex (roof) in the upper half of the embryos]. The embryos are ∼1.5 mm in diameter. After Chalmers et al., (2003).

a comparison with the development of mammalian skin is provided.

(a) Frogs X. laevis eggs begin to divide upon fertilization, increasing the number of blastomeres (cells produced via cleavage of the zygote), which continue to replicate synchronously during early embryogenesis. In the process of embryogenesis multiple cell divisions occur rapidly before the mass of cells thus created is ‘shaped’ into an embryo and finally a larva. At the 32-cell stage, the embryo is ball-shaped, with a onecell thick covering and an internal space (blastocoel) filled with ‘body fluid’. During the following division, in which the 64-cell embryo is formed, one cell leaves this singlecell layer becoming apposed on the internal compartment. It is this event in which a positional difference is first recognised (Chalmers, Strauss & Papalopulu, 2003), with the blastomeres in the original single-cell layer and the blastomere beneath it now called ‘outer’ or ‘superficial’ cells and ‘inner’ or ‘deep’ cells, respectively (Fig. 3). The number of deep cells increases thereafter, resulting in an embryo that is covered with a double layer of cells at the 128cell stage. The number of deep layers further increases to approximately five, dependent on region, at the 512-cell stage. Following this, the number of deep layers declines, with just one layer visible below the vertex (roof of embryo in Fig. 3) by the 1024-cell stage [the Nieuwkoop and Faber (NF), stage 8], when the embryo initiates drastic changes in its cell arrangements, known as gastrulation. At this stage, the embryo is ready to begin the process of developing an

embryonic epidermis using the two-cell (superficial and deep cell) -layered sheet. Until metamorphosis (a striking set of developmental changes that take place after hatching), the epidermis consists of just two layers, after which it becomes approximately five-layered, with the outer (apical) layer being shed (Itoh, Yamashita & Kubota, 1988). Scanning electron microscopy has shown that cilia begin to grow from the free surface of some ectodermal cells during the neural plate stage (NF-13) and then increase in number to the point where they are widely distributed over the surface of early tailbud stages (NF-18–22) (Kessel, Beams & Shih, 1974) (Fig. 4). Emergence of ciliated cells has also been detected at the inner (sensorial) layer early in the neurula stage (NF-15–16) (Chu & Klymkowsky, 1989). The ciliated epidermal cells persist, but the cilia regress by a process of resorption in NF-24 embryos and have mostly disappeared before hatching (NF-35). Studies on frog metamorphosis have mostly used two species as models: X. laevis and R. catesbeiana. Metamorphosis follows a programmed sequence of events: (i) premetamorphosis, in which larvae grow, (ii) prometamorphosis, in which larvae prepare for metamorphosis, and (iii) the metamorphic climax, in which larvae complete metamorphosis and become miniaturized frogs (froglets). Exact numerical staging of metamorphosis is species dependent, and can be described for X. laevis using the Nieuwkoop & Faber (1956) table and for R. catesbeiana using the criteria listed by Taylor & Kollros (1946) (TK) for R. pipiens. The progression of metamorphosis is largely regulated by thyroid hormones (THs). Circulatory levels of triiodothyronine (T3 ), the most potent form of TH, are undetectable in premetamorphosis, show a marginal increase



626

(A)

(A)

(B)

(B)

(C)

(C′)

(C)

(D)

Fig. 4. Non-keratinocyte-type embryonic epidermal cells in the Xenopus laevis embryo. The non-keratinocyte epidermal cells of X. laevis embryos were examined for morphological characteristics and distribution. (I) Ultrastructure of the epidermis as revealed by scanning electron microscopy. (A) Mucociliary epithelium including a ciliated cell, small secretory cell (*) and several large goblet cells. (B) Mucus-secreting goblet cells containing vesicles that will undergo exocytosis of their secretory contents, after which membrane remnants are left (arrowheads). (C) Lateral view of a ciliated cell with many apical kinocilia. (II) Fluorescence immunocytochemistry of the epidermis. (A) The X. laevis epidermis with a mixture of goblet and ciliated cells. The marker protein α-tubulin of ciliated cells is stained red, plasma membranes are labelled green with green fluorescent protein (GFP). Small secretory cells are indicated by asterisks. All other cells are goblet cells. (B) At higher magnification, membrane-GFP (green) reveals numerous secretory vesicles at the apical surface of goblet cells, and α-tubulin (red) in cilia. (C) Secretory vesicles in small cells (*) are stained by phalloidin (green) and a neighbouring ciliated cell contains α-tubulin (red). (C ) High magnification of a small secretory cell showing presumed secretory vesicles. (D) Diagram of cell types in the X. laevis epidermis. Goblet cells are the predominant cell type. Ciliated cells and small secretory cells (asterisks) are scattered throughout (after Hayes et al., 2007). The diameters of the silial clumps are estimated to be approximately 20 μm and the stage of embryos is considered to be around NF-25.

during prometamorphosis, rise rapidly around the beginning of metamorphic climax and reach a peak in the middle of metamorphic climax, then sharply decline to undetectable levels thereafter. It is reasonable to speculate that the wellrecognised regulation of mammalian skin physiology by TH, which includes the control of keratin expression, extracellular matrix deposition human hair growth and human hair follicle energy metabolism (Smith, Bahn & Gorman, 1989; Doshi, Blyumin & Kimball, 2008; Ramot et al., 2009; Paus, 2010; Vidali et al., 2013), reflects evolutionary conservation of the developmental effects of TH on anuran morphogenesis and metamorphosis (Kress, Samarut & Plateroti, 2009). (b) Mammals Fertilized mammalian eggs develop within a membrane to form blastocysts (∼32-cell stage in the mouse; generally

called the blastula stage), which is composed of the trophectoderm (∼20 cells) and the inner cell mass (ICM) (∼12 cells). The blastocyst adheres to the wall of the uterus (implantation), which is accompanied by decomposition of the egg membrane. The superficial membrane of an embryo at the blastocyst stage is thus replaced with the trophectoderm (TE). The ICM is composed of two cell populations: cells consisting of primitive endoderm (hypoblast) and those of primitive ectoderm (epiblast). It is the epiblast that gives rise to the embryo proper, which develops from foetus to infant, and matures to adulthood. The progenitor cell for the epidermis (the presumptive epidermal cell) is present in the epiblast. The key step for adult epidermis in early embryogenesis is the segregation of the epidermal ectoderm (primitive epidermal layer) from the neural ectoderm, which is differentially referred to as ‘superficial epithelium’ (SE) in the frog and TE in mammals. In humans, the embryonic


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epidermis is first established as a single-layered ectoderm, when the ectoderm and endoderm are defined in the ICM of the blastocyst at 7–8 days post-conception (Holbrook, 1983). Around 1 month post-conception, the epidermis becomes double-layered, the outer and inner layers being termed periderm and the basal layer, respectively. The latter is the layer of the epidermis proper and later functions as the epidermal basal layer. In mice, the original singlelayered epidermis becomes double-layered around day 8.5 (M’Boneko & Merker, 1988) and the periderm is shed shortly before birth. It is thought that some cells of the single-layered epidermis (‘periderm precursor cells’) detach from the basement membrane and migrate to the skin surface to form the periderm (Cui et al., 2007). The periderm functions as the epidermis during embryonic life, which in humans lasts until about 5 months post-conception (Carlson, 1990). The periderm terminates its function as the protective covering of the epidermis, and is sloughed off as a result of apoptosis (Polakowska et al., 1994) as soon as the underlying epidermal layers complete their differentiation (Holbrook, 1983). It is logical to assume that the single-layered epidermis of an early embryo is heterogeneous in the composition of its cell populations, consisting of at least two types of cell: basal precursor cells and periderm precursor cells (Cui et al., 2007). Both peridermal cells and basal cells, once established, behave as independent cell populations and are mitotically (A)

active at the double-layered stage of embryogenesis (Holbrook, 1983). The basal cells represent the progenitor cell type of adult germinative basal cells and generate intermediate cells approximately 2 months post-conception. The nomenclature of these cells relates to their location between the basal cell layer and periderm and also to their intermediate nature in the differentiation phase to the granular cells (Carlson, 1990). The intermediate cells proliferate actively and form a multi-layer. The cells of the uppermost layer differentiate into granular cells by undergoing incomplete keratinization around 5–6 months post-coitus, the time when the terminal differentiation is initiated, a process leading to the formation of the stratum corneum (Holbrook, 1983). Thereafter, the intermediate layer consists of spinous cells (Holbrook, 1983). Following these foetal developmental processes, developing human skin reveals histological structures essentially identical to adult-type skin structures present at birth. See online Appendix S2 in which embryonic development of the mamalian skin is compared with metamorphic development of the frog skin. Below, we describe adult frog and mammalian skin structure and function in more detail. (2) Epidermis Adult frog skin (Fig. 5A, B) consists of an outermost, ectoderm-derived and mucus-covered epidermis, generally consisting of 5–7 cell layers. On the surface is the thin (B) Co

E

Sp

M Ge

S

G

Me

De

M

G

C H

(C)

(D) E

Co

SG

Lu De

SW

Gr Sp

HF

H

Ba

Fig. 5. Morphology of adult Xenopus tropicalis and human skin. (A) X. tropicalis skin and (C) human skin, with white lines demarcating epidermis (E), dermis (De) and hypodermis/subcutis (H). Black arrows in A indicate margins of the stratum spongiosum (S) and stratum compactum (C). M, small mucus gland; G, large granular/poison gland; SG, human sebaceous glands; SW, sweat glands; HF, hair follicle. (B, D) Positions of the stratum germinativum (Ge), stratum spinosum (Sp) and stratum corneum (Co) of frog epidermis (B), and of the stratum basale (Ba), stratum spinosum (Sp), stratum granulosum (Gr), stratum lucidum (Lu) and stratum corneum (Co) of human epidermis (D). White arrow indicates a frog melanocyte (Me) at the dermal–epidermal junction. Scale bars in A and C, 300 μm; in B and D, 50 μm. Modified after Meier et al. (2013). Biological Reviews 89 (2014) 618–655 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society


628 stratum corneum, which is most often a monolayer of keratinized cells. Compared to the mammalian stratum corneum (Fig. 5D), the extent of keratinization is far less, resulting in greater gas and fluid permeability. It is this thin corneous layer that sets frog skin apart from that of fish, marking the evolutionary transition at which entirely aquatic species began to meet the demands of terrestrial living (Fox, 1986; Schempp, Emde & Wolfle, 2009). Beneath these cornified cells is the terminally differentiating epithelial layer of the stratum granulosum, a number of layers of stratum spinosum and the regenerative basal layer, the stratum germinativum (Fig. 5). As its name suggests, the mitotic activity of the latter layer serves to replenish and maintain the epidermal cell population. The basal cells are columnar in form, with morphology gradually changing as they migrate up through the layers of the epidermis, becoming flattened and typically squamous in nature, near the skin surface (Duellman & Trueb, 1994). The frog epidermis is home to a number of specialized cell types including sensory Merkel cells, mitochondria-rich flask cells, goblet cells (in the embryonic epidermis only), melanin-pigment-containing melanophores, and dendritic cells thought to be homologues of mammalian Langerhans

cells (Duellman & Trueb, 1994; Castell-Rodriguez et al., 1999; Hayes et al., 2007; Katz & Gabbay, 2010). The epidermis of aquatic vertebrates contains ionocytes specialized for regulating ion exchange and acid-base balance of body fluid (Brown & Breton, 1996). These cells have been described in the frog epidermis for over a century, as ‘goblet cells’ but also as ‘flask cells’. Flask cells (Whitear, 1976) have been reported to act as the cellular pathway for chloride movement across the amphibian skin (Katz & Larsen, 1984) (Fig. 6). They are now generally termed ‘mitochondria-rich cells’ and are specialized for chloride transport through dedicated channels (Larsen, 2011). The full complement of cell types present in frog skin is listed in Table 1, alongside their known mammalian counterparts. The stratum corneum of the amphibian epidermis is shed periodically in a zonal fashion, typically beginning at the head before continuing dorsally, and taking anywhere between 1 and 14 days for the process to be complete (Fox, 1986). Upon its increased keratinization, detachment from the underlying stratum granulosum begins as desmosomal connections are broken down, but the stratum corneum is not shed until cornification of the replacement layer is complete (Duellman

Table 1. Common frog skin cell types Cell type

Location

Key function(s)

References

Human skin equivalent

Keratinocytes

Epidermis

Duellman & Trueb (1994)

Keratinocytes

Dendritic cells

Epidermis

Protection Thermoregulation Osmoregulation Immune response

Langerhans cells

Goblet cells Flask cells

Epidermis (embryonic) Epidermis

Castell-Rodriguez et al. (1999) Hayes et al. (2007) Whitear (1976)

Mitochondria-rich cells

Epidermis

Merkel cells T cells B cells Nerve fibres

Epidermis Epidermis/dermis Epidermis/dermis Epidermis/dermis

Melanophores

Epidermis/dermis

Xanthophores

Dermis

Iridophores

Dermis

Mast cells

Dermis

Fibroblasts Myoepithelial cells

Dermis Dermis

Endothelial cells Adipocytes Stem cells

Dermis/hypodermis Hypodermis Unknown

Mucus secretion Water/ion transport Role in moulting Transport of Cl− , H+ , HCO3 − and organic molecules Mechanoreception Immune response Immune response Afferent connections to sensory (Merkel) cells, free nerve endings Pigmentation Camouflage Pigmentation Camouflage Pigmentation Camouflage Immune response Inflammatory response Connective tissue Surround glands aiding in secretory discharge Lining of blood vessels Energy storage Replication and maintenance of cell populations. Wound healing

— —

Brown et al. (1981) and Katz & Gabbay (2010)

—

Whitear (1989) Ramanayake et al. (2007) Horton et al. (1992) Koyama et al. (2001)

Merkel cells T cells B cells Nerve fibres

Bagnara et al. (1968)

Melanophores


—


—

Pelli et al. (2007)

Mast cells

Duellman & Trueb (1994) Rigolo, Almeida & Ananias (2008) Duellman & Trueb (1994) Toledo & Jared (1993a) —

Fibroblasts Myoepithelial cells


Endothelial cells Adipocytes Adult stem cells

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Fig. 6. Electron micrograph showing a secretory and a mitochondria-rich cell in a granular gland in adult Xenopus laevis frog skin. The secretory cell (sc) contains many secretory granules (sg) whereas the mitochondria-rich cell (mrc) is filled with mitochondria (m) and displays apical microvilli (arrows). Scale bar, 1 μm. After Kubota et al. (2006).

& Trueb, 1994). Often the frog will aid in the moulting process by mechanically removing sections of the moulting skin and ingesting them. As is common with higher vertebrates, the overall thickness of amphibian epidermis can vary considerably among species, as well as in different regions of the body. X. laevis is believed to have the thickest epidermis, with Bufo bufo possessing the thinnest (Brown, Grosso & De Sousa, 1981). This is accounted for by differences in the number of cell layers, as opposed to changes in the thickness of the stratum corneum. In addition, region and gender differences in the thickness of the integument have been noted (Greven, Zanger & Schwinger, 1995). The differences in skin thickness among frog species may be related to differences in mechanical load, e.g. during locomotion, in different habitats. The biomechanical features of frog skin have been studied in most detail for X. laevis, with regard to tensile strength, elasticity/stiffness, toughness, and maximal strain at break (Greven et al., 1995). Also, in contrast to the aquatic X. laevis, the toad Bufo bufo is adapted to humid terrestrial conditions. Because of this the epidermis is very thin to allow efficient water and gas exchange. In arid external conditions, Bufo bufo will move towards more humid environments such as caves to prevent excessive water loss. As the aquatic X. laevis likely requires a greater resistance to and control over transepidermal water movement, an increased epidermal thickness (through a greater number of keratinocyte layers, along with a thickened intra-epidermal tight junctional barrier, as opposed to stratum corneum thickening) may well facilitate this. Frog skin epidermis has been extensively characterised with regard to the expression of junctional complexes (Farquhar & Palade, 1965; Martinez-Palomo, Erlij & Bracho, 1971; Shahin & Blankemeyer, 1989; Nagel et al., 2002). In fact, a great deal of research characterized epithelial cell-cell communication and adhesion (including

barrier formation) in amphibian epidermis (Farquhar & Palade, 1965; Martinez-Palomo et al., 1971) decades before equivalent human research was well established (Kirschner et al., 2010). Research into human skin junctional complexes is now however, well established and an increasing body of evidence describes their functional importance in the mammalian epidermis [see Xu & Nicholson (2013) for a recent review on tight junctions and gap junctions in mammalian skin]. The basal and intermediate layers (stratum spinosum and stratum germinativum) of frog skin and to a lesser extent the stratum granulosum, are connected by a comprehensive network of gap junctions allowing the epidermis to act as a functional syncytium (Shahin & Blankemeyer, 1989). Desmosomes (maculae adherens) were also identified, with occurrence increasing from basal to apical including the stratum corneum (Shahin & Blankemeyer, 1989). Therefore, frog epidermis offers excellent opportunities to explore the full range of functions exerted by intraepithelial communication and compartmentalisation via gap junctions (Langlois et al., 2010; Becker, Thrasivoulou & Phillips, 2011; de Zwart-Storm et al., 2011) and tight junctions (Vockel et al., 2010; Haftek et al., 2011; O’Neill & Garrod, 2011) that are also relevant in human skin epithelium. (3) Epidermal mucus production In addition to the mucus produced by frog skin glandular structures, cells within the frog epidermis also produce mucus granules. Parakkal & Matoltsy (1964) identified differences between frog (R. pipiens) and mammalian epidermal layers in that cells of the mid differentiating layers of mammalian epidermis produce keratohyalin, whereas similar layers of the frog epidermis produce mucus granules instead. This mucus is secreted into intercellular spaces where it acts in a hydrophilic fashion to accumulate water, in this way maintaining skin moisture, reducing dehydration, and allowing efficient cutaneous gas exchange, which, as will be discussed later, are crucial functions of the frog integument (Parakkal & Matoltsy, 1964). (4) Dermis Beneath the epidermis and separated by a collagenous basement membrane is the substantially thicker dermis, of mesodermal origin (Fig. 5). In both frogs and mammals, the dermis is subdivided into two compartments: the stratum spongiosum and stratum compactum in frogs, being respectively the papillary and reticular dermis in humans (Felsemburgh et al., 2009). The stratum spongiosum consists of loosely packed collagen and elastin fibres and in addition contains numerous alveolar glands, chromatophores, nerve fibres and blood vessels that supply the epidermis. The stratum compactum shows a much greater density of orthogonally arranged (longitudinal and transverse) collagen fibres, which anchor the dermis to the underlying muscle. The longitudinal fibres form the boundaries of wide tracts that are assumed to provide pathways for the migration



630 of cells such as melanophores (Zhu, 1987; Denefle, Zhu & Lechaire, 1993; Greven et al., 1995). The mucus and granular glands of the anuran integument are embedded predominantly within the stratum spongiosum, with ducts protruding into and through the epidermis. Despite the bulk of these structures residing within the dermis, their developmental origins lie in the ectoderm rather than in the mesoderm. The granular (poison) glands are consistently larger in size than the mucus glands, and whereas the latter are surrounded by a cuboidal epithelium only, the encapsulating connective tissue of their larger counterpart is encircled with smooth muscle cells that compress the gland and thus aid in the ejection of granular secretions through the lumen (Mills & Prum, 1984). The dermis is also richly supplied by capillaries, which, as will be described later, provide an important route for cutaneous gas exchange. Frog mucus glands have historically been of interest in terms of their evolutionary association with the human sweat gland (Davis & Hadley, 1979), their thin, watery secretions aiding in thermoregulation and reducing susceptibility to desiccation. Consequently, they are particularly active in terrestrial species frequently exposed to direct sunlight (Lillywhite & Licht, 1975). By contrast, the granular poison glands produce thick, milky secretions that range from mildly noxious to extremely toxic (Mills & Prum, 1984). Chromatophores, or pigment cells, derive from the neural crest and produce the diverse hues of the various anuran species, serving as camouflage and protection from predators and also signalling to possible enemies that toxic poisons may be secreted. Three types of chromatophore occur in the dermis: xanthophores, iridophores and melanophores, together forming the dermal chromatophore unit (Bagnara, Taylor & Hadley, 1968). These cell types are considered in some detail in Section IX. (5) Hypodermis (subcutis) As in mammalian skin, subdermal regions of frog skin are sometimes referred to as the subcutis or tela subcutanea. In anuran species, they consist of a thin layer of vascularised loose connective tissue, generally containing a few adipocytes and providing the covering of lymph sacs (Toledo & Jared, 1993a). In some species, such as Hyperolius nasutus and Hyperolius viridiflavus, they may also contain iridophores (Kobelt & Linsenmair, 1986; Toledo & Jared, 1993a), and recent studies have revealed certain members of the aquaporin family of water-transporting proteins in this layer in Hyla chryoscelis (Pandey et al., 2010). (6) Innervation In view of the highly complex structure and functions of frog skin, it is not surprising that its innervation is extremely intricate. In addition to the extensive motor innervation of smooth muscle cells involved in glandular and vascular control, there is a vast network of sensory fibres possessing

either free endings or synaptically transferring signals from receptor cells towards the central nervous system. To illustrate this complexity, the innervation of the frog skin will be discussed in relation to mechano-, temperature and magnetic field receptors, as well as to its vascularization and exocrine activities. (a) Lateral line system The lateral line system of amphibians consists of numerous epidermal mechanoreceptors distributed over the whole body surface. Apically, a receptor cell possesses a group of stereocilia and one kinocilium, which protrude into a jelly-like cupula that extends into the surrounding fluid. Displacement of the cupula bends the cilia to stimulate receptor activity. Two large, afferent fibres innervate one group of receptor cells (neuromast). The fibres branch repeatedly to supply multiple neuromasts (Winklbauer, 1989), their sensitivity increasing with the number of neuromasts they innervate (Mohr & Görner, 1996). Interestingly, in Xenopus laevis tadpoles, kinocilia of early neuromasts are not embedded in a cupula but project out from the skin surface, to detect water movements (Roberts et al., 2009). (b) Merkel cells Merkel cells are specialized sensory cells that occur in small groups around the openings of the cutaneous exocrine gland ducts. The physiological, morphological and trophic characteristics of the Merkel-cell-neurite complexes in frog skin have been studied extensively in X. laevis (Mearow & Diamond, 1988) and their ultrastructure in the toad Bufo icterus (de Brito-Gitirana & Azevedo, 2005). Sensory axons contact Merkel cells via morphologically specialized contacts involving reciprocal synapses. Possibly, Merkel cells enhance or even induce the excitability of other mechanosensitive nerve endings in the skin (Mearow & Diamond, 1988). It has been suggested that in the toad Bufo bufo their functioning depends on anterior pituitary gland activity (Budtz & Zaccone, 1990). (c) Thermoreceptors Another extensively studied receptor type in the frog skin is the specific thermoreceptor, which belongs to an electrophysiologically distinct class of cutaneous receptors with a morphological substrate (free nerve endings) and plausible transduction mechanism (electrogenic Na+ pump). Frog cutaneous receptors respond only to cooling. Thermal stimulation of frog skin produces a discharge in afferents in the dorsocutaneous nerve. Antidromic occlusion experiments have demonstrated the relative insensitivity of these receptors to tonic mechanical stimulation (e.g. Holloway, Ramsundar & Wright, 1976). Sympathetic modulation of thermal sensitivity is mimicked by experimental administration of adrenalin and noradrenalin and of the adrenergic agonist, ephedrine (Spray, 1974). For a comprehensive review of


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the morphology, functioning and sympathetic innervation of frog temperature receptors, see Spray (1986). (d) Rohon-Beard neurons A special kind of innervation of the frog skin is provided by the Rohon-Beard neurons in the spinal cord, which, together with extramedullary neurons lying outside the cord, form a sensory neurite plexus on the basal lamina of trunk skin in Xenopus laevis tadpoles, and innervate the underlying epidermal cells through holes in the lamina (Roberts & Taylor, 1982; Taylor & Roberts, 1983). RohonBeard neurons show substance-P-like immunoreactivity in their somata and efferents. They detect weak touch stimuli to the body in response to which they initiate swimming (Clarke et al., 1984). (e) Frontal organ Another interesting type of innervation of the frog skin concerns the frontal nerve, which interconnects the frontal organ with the epiphysis (pineal gland) and the brain. The frontal organ is an outgrowth of the pineal gland, and has been reported to be sensitive to magnetic fields (DiegoRasilla, Luengo & Phillips, 2010). In Rana esculenta the frontal nerve forms, either on its left or its right side, a unilateral branch that runs into the dermis. Therefore, the lateral nerve may represent a pathway interconnecting the skin and the brain and/or the frontal organ (Guglielmotti, Fiorino & Sada, 1995). (f ) Vascular system The intricate cutaneous blood flow in frogs is under complex nervous control (Smith, 1976). Stimulation of cranial nerve I, the vagal ganglion, sympathetic ganglion 1 and sometimes sympathetic ganglion 2 increases cutaneous vascular resistance, i.e., the resistance to flow that must be overcome to push blood through the circulatory system of the skin. The cutaneous vasculature receives two types of vasomotor input: from sympathetic nerves that are probably adrenergic, and from other nerves that are non-adrenergic/noncholinergic and do not use ATP as a transmitter. Alpha-adrenoceptors mediate the constrictor responses to sympathetic nerve stimulation and catecholamine administration. Beta-adrenoceptors also occur, but their role in vascular control seems minor (Malvin & Riedel, 1990). More recently it was found that, in the toad Bufo marinus, neurally derived nitric oxide (NO) regulates vascular tone in cutaneous arteries (Jennings & Donald, 2008). (g) Exocrine gland innervation Both mucus and granular glands in frog skin (Skoglund & Sjöberg, 1977) receive exclusively adrenergic innervation, but the distribution of nerve terminals differs between the two types of gland. In the mucus gland, terminals end at a distance of about 0.5 mm from the basement membrane and never

enter the gland parenchyma. In the granular gland, axon terminals are located between smooth muscle cells and also form direct contacts with secretory cells, yet never occur outside the basement membrane (Sjöberg & Flock, 1976; Davis & Hadley, 1979). A detailed study of the calcium-dependent mechanism underlying exocrine chloride secretion by frog skin was made in R. esculenta (Bjerregaard, 1989).

V. CHARACTERISTIC FEATURES AND NOVEL ADAPTATIONS OF FROG SKIN Although many of the functions of frog skin can also be found in mammalian skin, at least in rudimentary form, frog skin excels in a number of cutaneous properties that are considered as ‘trademark’ features, the evolutionary roots of which can be traced back to fish skin (Rakers et al., 2010). They concern physiological actions to minimise water loss, water uptake by special membrane channels (aquaporins, AQPs), gas exchange, release of predator repellents, acquisition of defence alkaloids, and skin adaptations to hibernation. These characteristic features and novel adaptations of frog skin are discussed below. (1) Minimising water loss Most frogs lack resistance to cutaneous water loss, as moist skin is necessary for proper transcutaneous respiration (Chew, 1961). Therefore they are exposed to the severe threat of evaporative water loss (EWL), from which most frogs try to protect themselves behaviourally through being primarily nocturnally active, having a fossorial (burrowing) behaviour, and/or residing close to a water source. Some species can even use a ‘seat pouch’ on the belly to absorb moisture from plants or soil through capillary action. Surprisingly, frogs are found not only in watery environments but also in a great variety of terrestrial habitats, including deserts (Duellman & Trueb, 1994). These animals have acquired special physiological adaptations, such as in the arid-adapted African treefrog Chiromantis petersi. This frog possesses numerous layers of iridophores (light-scattering skin chromatophores), rather than a one-cell-thick layer, as in most frogs. In C. petersi skin, the dorsum, sides and throat regions can have up to five layers of iridophores, which may substantially protect against EWL (Drewes et al., 1977). Some frog species reveal other adaptations to arid conditions. They possess skin glands that secrete lipids onto the skin surface, which are then wiped across the body, thus producing a lipid-like ‘wax’ film that strongly reduces EWL (Arney & Grigg, 1995; Hillyard, 1999; Barbeau & Lillywhite, 2005). Frogs of the phyllomedusine family have even been described as ‘waterproof’, displaying low levels of EWL characteristic of certain desert reptiles, and representing only 5–10% of levels measured in aquatic and terrestrial fossorial frogs subjected to a desiccating environment (Shoemaker et al., 1972). In other frog species



632 however, this wax-wiping behaviour is less successful. In the Indian Polypedates maculatus, for instance, wiping behaviour reduces EWL by only 50% compared to water evaporation from a free water surface (Lillywhite et al., 1997). Such species possess other behavioural adaptations to limit EWL, such as postural changes to reduce the surface area of sunexposed skin. Once lipid secretions have been applied (wiped) across the body, these frogs become relatively immobile, also preventing disruption of the freshly applied lipid barrier.

outside the kidneys of these species. Furthermore, in contrast to the hylid frog, pelvic skin did not reveal any AVTassociated changes in water movement in R. japonica or R. nigromaculata. Conversely, in the same study, the skin of the entirely aquatic X. laevis, which needs to minimise water uptake, was entirely unresponsive to AVT (Ogushi et al., 2010). These investigations highlight the evolutionary mechanisms that have allowed the adaptation of different frog species to their respective environments. (4) Gas exchange

(2) Cocoon formation A particular mechanism by which certain frog species survive the harsh winter period is by overwintering in a self-made cocoon. The desert-dwelling Cyclorana alboguittatus, C. platycephaluts and C. australis, form cocoons from transparent cell layers derived from the stratum corneum, which covers the entire body (with the exception of the nostrils) and reduces EWL by over sevenfold, allowing the animals to remain buried in the soil for many months (Lee & Mercer, 1967). Formation and constitution of a cocoon can be very complicated. In Lepidobatrachus ilanensis and Smilisca baudini, for instance, layers of the stratum corneum with a thickness of up to 60 cells are formed, separated from each other by sub-corneal spaces filled with mucus (Toledo & Jared, 1993a). As the number of corneal layers increases, so the degree of EWL decreases (McClanahan, Ruibal & Shoemaker, 1983). (3) Water uptake by aquaporins Unlike higher vertebrates, most amphibians do not physically drink water through the mouth. Instead, water uptake is controlled through the skin. Especially in semiaquatic and terrestrial frogs, major water movements occur via the ventral, pelvic skin. This active process is controlled largely via channels formed by aquaporins (AQPs), a family of water and glycerol transporters (aquaglyceroporins; reviewed in Verkman & Mitra, 2000). Frogs share many AQPs with mammals yet anuran-specific AQPs have also been identified: AQPa1 and AQPa2, with AQP-h2 and AQPh3 as subtypes (Suzuki & Tanaka, 2010), and AQP3 with subtype AQP-h3BL in hylid frogs (Akabane et al., 2007) and subtype AQP-x3 in X. laevis (Mochida et al., 2008). Frog AQPs are tissue- and species-specific in terms of both their expression and regulation (Suzuki et al., 2007; BouryJamot et al., 2009). For example, in the pelvic epithelium of the arboreal frog, Hyla japonica, both AQP-h2 and AQP-h3 are stimulated by the anuran homologue of mammalian vasopressin, arginine-vasotocin (AVT) (Hasegawa et al., 2003), a process apparently brought about by AVT-induced relocation of AQP-h3 from the cytoplasm to the apical plasma membrane (Suzuki & Tanaka, 2010). Likewise, in semiaquatic ranid species Ogushi et al. (2010) found that skin from ventral hindlimbs responded to AVT by increasing AQP-h3 expression in R. japonica, R. nigromaculata and R. catesbeiana; they could not detect expression of AQP-h2

Frog skin displays a high permeability to oxygen and carbon dioxide. More than a century of research has shown the importance of cutaneous excretion of carbon dioxide, which quantitatively dominates that of exhalation (Piiper, 1982; Jørgensen, 2000). The ability of the skin to control oxygen absorption varies among species, environments and temperature (Malvin & Hlastala, 1989; Boutilier et al., 1997). A striking example of this capacity is presented by overwintering aquatic ranid frogs, which alter cutaneous oxygen uptake in response to a change in external partial oxygen pressure. This property, together with other physiological adaptations, aids these animals in their struggle for survival under harsh environmental conditions (reviewed by Tattersall & Ultsch, 2008). (5) Predator repellents Various poisonous compounds are secreted from granular glands in frog skin. These glands are generally fewer in number than mucus glands, and are located in distinct regions as dictated by the defence requirements imposed upon the animal by the environment. Terrestrial amphibians may display clusters of granular glands in the head and shoulders whereas aquatic species such as X. tropicalis have an almost even distribution on both dorsal and ventral skin (Toledo & Jared, 1993b). Poison glands are also the source of the extremely broad array of bioactive peptides isolated from frog skin, a phenomenon that will be discussed in detail in Section VII. (6) Chemical defence alkaloids A remarkable property of some anurans is the ability to take up external lipid-soluble alkaloids into their skin glands, which can then serve as a defence against micro-organisms and predators (Daly, 1998). The occurrence of over 800 alkaloids in amphibians seems phylogenetically restricted; these compounds have been mainly reported in one speciesrich lineage of neotropical poison-dart frogs (Dendrobatidae), including the South-American bufonid Melanophryniscus moreirae, Madagascan mantellids (e.g. Mantella baroni and M. betsileo) and the Australian myobatrachid Pseudophryne coriacea (for details see Daly, Spande & Garraffo, 2005). Dietary specialization may be an important aspect in the evolution of sequestered alkaloids in frogs. Ants were assumed to be a primary source of these alkaloids (Jones


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& Blum, 1983; Numata & Ibuka, 1987; Blum, 1992), but more recently several alkaloids in the frog skin have been suggested to be produced by oribatid mites. For instance, in the dendrobatid poison frog Oophaga pumilio 25 5,8-disubstituted or 5,6,8-trisubstituted indolizidines, one 1,4-disubstituted quinolizidine, three pumiliotoxins and one homopumiliotoxin have been found (see Saporito et al., 2007) and in Eleutherodactylus iberia, a species of miniaturized frogs of the Eleutherodactylidae, six pumiliotoxins and two indolizidines appear to be derived from various oribatid mite species. This has raised the idea that in these amphibians, miniaturization and specialization to small prey may have favoured the acquisition of dietary skin alkaloids (Rodríguez et al., 2011).

VI. (NEURO-)ENDOCRINOLOGY OF FROG SKIN Over the preceding two decades it has been established that human skin functions as a neuroendocrine organ, locally producing a range of hormones (i.e. hypothalamic neurohormones, pituitary hormones, steroid hormones and thyroid hormones) and possessing receptors for neuropeptides and neurotransmitters classically expressed in the central nervous system and endocrine organs (Slominski & Wortsman, 2000). This cutaneous (neuro)endocrine system plays a vital role in maintaining skin and whole-body homeostasis (Slominski et al., 2007). Such a hormonal function has also been demonstrated for frog skin (Bagnara, Taylor & Prota, 1973; Vaudry et al., 1999; Takada & Hokari, 2007). Whereas frog skin has long been known as a target for various hormones (e.g. the action of noradrenalin and αMSH on skin melanophores), hormonal signal trafficking in the frog skin is not unidirectional, as three hypophysiotropic compounds, TRH, sauvagine and skin peptide tyrosinetyrosine (SPYY) are produced in frog skin (Mor et al., 1994b; Vaudry et al., 1999). SPYY, in very much the same way as its paralogue, neuropeptide Y (NPY), acts as an α-MSH releaseinhibiting factor (Fig. 7) (Danger et al., 1986; Verburg-Van Kemenade et al., 1987b; Chartrel et al., 1991; Mor et al., 1994b). Sauvagine, like its paralogues corticotropin-releasing hormone (CRH) and urotensin I/urocortins, stimulates adrenocorticotropin and α-MSH secretion (Lederis et al., 1982; Fryer, Lederis & Rivier, 1983; Rivier et al., 1983; Verburg-Van Kemenade et al., 1987a). Thanks to the use of techniques such as reverse-phase high-performance liquid chromatography (HPLC) combined with electrospray mass spectrometry, an even higher number of hormonally active factors have recently been isolated from frog skin. Below we review skin hormonal factors and their (putative) functions in more detail.

Fig. 7. Transduction of various physiological environmental stimuli to the endocrine melanotrope cell of Xenopus laevis controlling the secretion of α-melanophore-stimulating hormone (α-MSH), via neurohemal nerve terminals in the pars nervosa and from synaptic terminals in the pars intermedia of the pituitary gland. The melanotrope cell also releases auto-excitatory messengers (curved arrows). 5HT, 5-hydroxytryptamine (serotonin); A, arginine-vasopressin receptor; ACh, acetylcholine; AVT, arginine-vasotocin; β, βadrenergic receptor; BDNF, brain-derived neurotrophic factor; C, calcium-sensing receptor; C1, corticotropin-releasing factor receptor 1; C2, corticotropin-releasing factor receptor 2; CRF, corticotropin-releasing factor; D2 , dopamine D2 receptor; DA, dopamine; Ga , GABAA receptor; Gb , GABAB receptor; GABA, γ -aminobutyric acid; M, mesotocin receptor; M1, muscarinic M1 receptor; ME, metenkephalin; Me, metenkephalin receptor; MT, mesotocin; NA, noradrenalin; NPY, neuropeptide Y; P, P75NTR receptor; PACAP, pituitary adenylate cyclaseactivating polypeptide; T, tropomyosin receptor kinase B; T3, thyrotropin-releasing hormone receptor 3; TRH, thyrotropinreleasing hormone; Ucn1, urocortin 1; V1, VPAC1 receptor; Y1, NPY Y1 receptor. Modified after Roubos, Scheenen & Jenks (2005).

linked with temporal changes in the expression of certain AMPs (Ohnuma, Conlon & Iwamuro, 2009). TH signalling is regulated via thyroid hormone receptors (TRs) acting as heterodimers with constitutively expressed 9-cis retinoic acid receptors. This complex binds to specific thyroid response elements in target genes to exert transcriptional control (Evans, 1988). During amphibian metamorphosis striking changes occur in the morphogenesis and differentiation of the epidermis. Simultaneously with these changes, the adulttype 63 kDa keratin gene is strongly upregulated. These processes are unique because they are controlled by T3 , which directs upregulation of activating protein-2 in the epidermis (Mathisen & Miller, 1989; French et al., 1994).

(1) Thyroid hormones

(2) Thyrotropin-releasing hormone

As briefly discussed in Section IV, interest in TH signalling has focused on the hormone’s role in larval metamorphosis (Mathisen & Miller, 1989; Yoshizato, 2007) and has been

TRH was initially isolated from ovine (Burgus et al., 1970) and porcine (Nair et al., 1970) hypothalamic extracts on the basis of its ability to stimulate TSH release. Importantly,



634 the structure of TRH is entirely conserved from fish through amphibians to mammals (Galas et al., 2009), making amphibians an excellent model for studying TRH activity and mechanisms of action (Yasuhara & Nakajima, 1975). The skin of certain frog species (X. laevis, R. ridibunda, Bufo orientalis) produces extremely high amounts of TRH (Yasuhara & Nakajima, 1975; Giraud et al., 1979), with R. pipiens skin producing approximately twice as much TRH as its hypothalamus (Jackson & Reichlin, 1977). The high plasma concentrations of TRH found in these frog species [in R. pipiens: 1000–10000 times the concentration in mammalian skin (Jackson & Reichlin, 1979); see also Bolaffi & Jackson, 1982] is likely the result of catecholamine-stimulated release of this skin-derived TRH into the circulation. The notion that TRH could be released from the skin into the circulation adds to the credentials of frog skin acting as a true endocrine organ, e.g. exerting regulatory control over pituitary gland secretory activity (Vaudry et al., 1999). Production of TRH by cells of amphibian poison glands implies a dual endocrine/exocrine function in frog skin, which has similarly been described for TRH in human skin (Bodo et al., 2010). Hypothalamic TRH has been considered as a multipotent hypophysiotropic factor (Galas et al., 2009) and, in addition to TSH, stimulates the release of prolactin (PRL; Leong, Frawley & Neill, 1983), growth hormone (Szabo et al., 1984), vasopressin and insulin in mammals (Wilber & Utiger, 1968). In both R. ridibunda (Tonon et al., 1980, 1983; Lamacz et al., 1987; Galas et al., 1998) and X. laevis (Verburg-Van Kemenade et al., 1987a) TRH acts as an α-MSH-releasing hormone (Fig. 7), playing a key role in the process of frog skin pigment adaptation. Indeed, Smith (1916) first showed that hypophysectomy of frog tadpoles induces skin bleaching. These studies are of profound interest for mammalian skin research and TRH has since turned out to be a potent stimulator of human hair growth, hair pigmentation and of mitochondrial activity and biogenesis in human skin epithelium (Knuever et al., 2012; Vidali et al., 2013) (Bodó et al., 2010; Gaspar et al., 2010, 2011). These recent discoveries likely reflect the fact that mammalian skin has conserved not only the production of many peptides also generated in amphibian skin, but still exploits at least some of their evolutionarily ‘ancient’ functions (such as stimulation of pigment movement and growth). The existence of important roles for frog (neuro)hormones produced by and acting in the mammalian (including human) skin has only recently become recognised. (3) Prolactin In addition to THs, the influence of PRL on amphibian metamorphosis has been the subject of many studies, with conflicting results. In particular, administration of mammalian PRL has been reported to have antimetamorphic effects and to extend the tadpole phase of the life cycle of many amphibian species (St Germain et al., 1994; Berry et al., 1998a; Berry, Schwartzman & Brown, 1998b) as well as

neutralizing the effects of TH when added to cultured tadpole tissues (Eliceiri & Brown, 1994). PRL also inhibits limb morphogenesis induced by T3 (Tata, Kawahara & Baker, 1991). However, more recent publications have questioned the role of PRL as the ‘juvenile hormone’ involved in frog larval development (Huang & Brown, 2000) and it has been suggested that PRL has a dual role in amphibian metamorphosis: acting to modulate TH signalling but also inducing the development of adult-type features of the epidermis such as Na+ transport ability (Takada & Hokari, 2007). Indeed, PRL influences osmoregulation in fish (Sakamoto & McCormick, 2006) and Takada & Hokari (2007) demonstrated the ability of PRL to stimulate passive (via the epithelial Na+ channel, ENaC) and active (via the Na+ /K+ ATPase) Na+ transport across the skin of adult bullfrogs. Possibly, the hormone plays similar roles in other vertebrates. (4) Cholecystokinin Cholecystokinins (CCKs) are neuroendocrine peptides produced in the gastrointestinal tract and central nervous system of mammals, and several CCKs have also been identified in these organs in amphibians, especially in R. catesbeiana and X. laevis, but not in skin. However, a CCK isoform with the same sequence as CCK of R. catesbeiana was found in skin secretory material of the dark-sided tropical frog, R. nigrovittata (Liu et al., 2007). This suggests that CCK may play a role in the communication between brain, gastrointestinal system and skin. (5) Insulin-releasing factors Some peptides from the skin of Gunther’s frog, Hylarana guntheri stimulate the release of insulin from rat BRINBD11 beta-cells. The most potent peptide in this respect is brevinin-2GUb; other peptides with weaker insulin-releasing activity belong to the brevinin-1 (two peptides), brevinin2 (two peptides) and temporin (three peptides) families. Administration of brevinin-2GUb to mice significantly improved their glucose tolerance, demonstrating that the peptide or its derivatives has the potential for treatment of type 2 diabetes (Conlon et al., 2008b). Indeed, many frog-skinderived peptides are known to stimulate insulin release (e.g. Abdel-Wahab et al., 2008; Conlon et al., 2008b; Mechkarska et al., 2011), with the recent combination of HPLC and mass spectrometry of noradrenalin-stimulated skin secretions of Lithobates catesbeianus, X. laevis and Silurina epitropicalis further leading to the identification and characterization of several novel insulin-release-stimulating peptides: brevinin-1CBb, ranatuerin-2CBd (Mechkarska et al., 2011) and caerulein precursor fragments (Srinivasan et al., 2013). (6) Pheromones Amphibian tadpoles reveal rapid and sustained behavioural inhibition upon exposure to chemical cues of predation. Ranid tadpole skin releases an alarm pheromone into the medium upon predator attack, signalling the predator’s


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presence to conspecifics. The pheromone consists of two components with distinct biophysical properties that must be combined to elicit the behavioural response. In addition, the pheromone rapidly suppresses the activity of the hypothalamo-pituitary-adrenal (HPA) axis, as evidenced by a decrease in the corticosterone titre. This finding seems to be the first evidence for an aquatic vertebrate prey actively secreting an alarm pheromone in response to a predator’s attack (Fraker et al., 2009).

VII. FURTHER SECRETORY ACTIVITIES OF FROG SKIN: AMPS As established by V. Erspamer and co-workers (Anastasi, Erspamer & Bucci, 1971; Erspamer, 1971, 1984; Erspamer et al., 1981; Melchiorri, 1985; Erspamer, Falconieri Erspamer & Cei, 1986; Severini et al., 2002), secretions from the anuran integument contain an extraordinarily varied source of active compounds, ranging from biogenic amines (e.g. serotonin), complex alkaloids and steroids (Clarke, 1997) to polypeptides with differing antimicrobial or pharmacological activities [e.g. tachykinins, bradykinins, caeruleins, TRH, bombesins, the trypsin/thrombin inhibitor BSTI, Bv8 (Bombina variegata protein; molecular mass 8 kDa) and opioid peptides], many of which have counterparts in the mammalian brain and gastrointestinal tract (Simmaco, Kreil & Barra, 2009). This has led to the concept of the existence of a brain-gut-skin peptide triangle (Erspamer et al., 1981). The sequences of frog skin peptides are so diverse, even in closely related species, that they can be used to complement classical phylogenetic analyses and/or to revisit amphibian taxonomy (Conlon et al., 2008a, 2010). Table 2 lists key compounds secreted by the skin of anuran Amphibia, with antimicrobial or pharmacological effects. The equivalent peptides in mammalian tissues are also indicated. Below we will focus on antimicrobial peptides (AMPs) in more detail. AMPs are essential compounds fulfilling the conserved, nonspecific innate immune response of most multicellular organisms, providing a fast-acting shield against microbial infections (Boman, 1995; Zasloff, 2002). This protection is required, in the case of vertebrates, before the adaptive immune system is activated (Ganz, 2003). Despite the diversity in their sequences and conformations, most AMPs share similar features: (i) small size (12–50 amino acid residues), (ii) an overall net positive charge at neutral pH, and (iii) an amphipathic structure (α-helix, β-sheet or both) following interaction with a biological membrane (Shai, 2002). The initial interactions of these molecules with target cells is typically mediated by electrostatic binding. In plasma membranes of eukaryotic cells, the outer leaflet is mainly composed of neutral phospholipids, carrying little or no electric charge. By contrast, both leaflets of bacterial cell membranes contain a high proportion of acidic phospholipids (e.g. phosphatidyl glycerol, cardiolipin) conferring a net negative charge to the surface. These differences are believed to underlie the preferential binding

and activity of AMPs towards microbial cells (Shai, 1999; Zasloff, 2002). However, before reaching the microbial plasma membrane, peptides need to cross a cell wall that in Gram-negative bacteria is surrounded by the anionic outer membrane composed mainly of lipopolysaccharide (LPS or endotoxin) (Raetz & Whitfield, 2002). Mode of action studies have indicated that the majority of AMPs (especially those with an α-helix structure) physically permeate the bacterial plasma membrane either via transmembrane pore formation (barrel stave mechanism) or via membrane disintegration in a detergent-like manner (carpet-like mechanism) that can involve the generation of ‘toroidal’ pores, channel aggregates or more complex structures (Matsuzaki, Murase & Miyajima, 1995; Ludtke et al., 1996; Hancock & Rozek, 2002), depending on the length and the sequence of the peptide. Overall, this results in the collapse of transmembrane electrochemical gradients, leakage of metabolites and eventually cell lysis and death (Hancock, 1997; Brogden, 2005). Nevertheless, there is convincing evidence that membrane perturbation is not the only mechanism of microbial killing. Indeed, some peptides can penetrate or pass the plasma membrane and subsequently inhibit various vital functions (e.g. membrane septum formation, cell wall/nucleic acid/protein synthesis) or alter enzymatic activity (Brogden, 2005). This multiple targeting and physical disruption of fundamental physiological cell structures (e.g. the plasma membrane) makes microbes less likely to develop resistance to AMPs compared to conventional antibiotics many of which act via receptor-mediated mechanisms (Yeaman & Yount, 2003). AMPs have different specificities: some kill only bacteria (a specific strain or a wide range of bacterial species), some kill only fungi, while others are active against a broad spectrum of pathogens such as bacteria, yeasts, fungi, protozoa and viruses, sometimes also being toxic to mammalian cells. This functional diversity exists as a result of the varying biophysical parameters of AMPs (e.g. charge distribution, length, amphipathy, oligomeric state), in addition to the chemical nature of the target cell surface itself, which can also affect cell selectivity. Although AMPs were initially discovered on the basis of their antibiotic activity (to date, more than 1000 native AMPs are known), additional biological properties that assist in modulating the host immune system have been detected in recent years: stimulation of chemotaxis, suppression of pro-inflammatory cytokine production, promotion of angiogenesis, mast cell activation (Chen et al., 2005), modulation of cellular differentiation pathways and wound healing (Yeung, Gellatly & Hancock, 2011). Moreover, some AMPs neutralize the toxic effect of the microbial LPS (e.g. PR-39, LL-37, HNP1–3; Bowdish et al., 2005) or possess anticancer activity (Yeung et al., 2011). Owing to these findings, AMPs are now more properly referred to as host-defence peptides (Yeung et al., 2011). Among the natural sources of AMPs, amphibian skin is one of the richest storehouses, from which many hundred distinct forms have been isolated (Fig. 8) (for a comprehensive


Erspamer et al. (1979) Conlon et al. (2009) Conlon et al. (2009)

Criana

Alytes

Ascaphus

Angiotensin

Alyteserins

Ascaphins

Erspamer et al. (1985) Anastasi, Erspamer & Bertaccini (1965) Nakajima et al. (1979) Conlon (2011) Conlon et al. (2008b) Conlon et al. (2008a) Conlon et al. (2010) Anastasi, Erspamer & Endean (1968) Anastasi et al. (1970) Anastasi et al. (1970) Montecucchi, Falconieri Erspamer & Visser (1977) Anastasi et al. (1969) Mor & Nicolas (1994) Mor & Nicolas (1994) Kreil et al. (1989) Simmaco et al. (1993) Conlon (2011) Conlon (2008) Conlon et al. (2008a)

Phyllomedusa Rana Heleophryne Rana

Hylarana Odorrana

Leptodactylus Xenopus Hylambates

Phyllomedusa Phyllomedusa

Phyllomedusa Phyllomedusa Rana

Lithobates Phelophylax Odorrana

Phyllocaeruleins Dermaseptins

Dermorphins Deltorphins Esculentins

Caeruleins

Brevinins

Hyla

Mignogna et al. (1993)

Bombina

Bradykinins

Anastasi et al. (1971) Anastasi, Erspamer & Bucci (1972) Anastasi, Erspamer & Endean (1975) Barra et al. (1985) Yasuhara, Ishikawa & Nakajima (1979) Simmaco et al. (2009)

Bombina Alytes Litoria Phyllomedusa Rana Bombina

Bombesins Bombesin Alytesin Litorin Phyllolitorin Ranatensin Bombinins/ Bombinin-like peptides Bombinins H

References

Representative genera

Peptide family

— Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ Enkephalin — Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ — — —

— — —

Gastrin/cholecystokinin

GRP Neuromedin B Neuromedin C — — Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ Cathelicidin LL-37/hBD1-43, RNase 7, psoriasin∗ Bradykinin/kalidin — — Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ — —

Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗

Angiotensin

Mammalian equivalent

Table 2. Peptides with comparable pharmacological or antimicrobial functions in frog skin and mammalian tissues

— Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) Hughes et al. (1975) — Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) — — —

Gregory & Tracy (1964) and Anastasi, Erspamer & Endean (1967) — — —

McDonald et al. (1978, 1979) Minamino, Kangawa & Matsuo (1983) Minamino, Kangawa & Matsuo (1984) — — Bernard & Gallo (2011) and Glaser et al. (2005) Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) Elliott, Horton & Lewis (1960) — — Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) — —

Skeggs, Kahn & Shumway (1952), Skeggs et al. (1955) and Lentz et al. (1956) Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b)

References

636



Amiche et al. (2008) Amiche et al. (2008) Conlon (2011) Conlon (2008) Montecucchi et al. (1981) Mor et al. (1994b) Hoffmann (1988) — Anastasi et al. (1977) Erspamer and Anastasi (1962) Erspamer et al. (1966) Simmaco et al. (1996) and Mangoni (2006) Conlon et al. (2008b) Conlon et al. (2010) and Mangoni & Shai (2009) Yasuhara & Nakajima (1975) and Roseghini et al. (1989) Araki et al. (1973, 1975)

Phyllomedusa

Phyllomedusa

Rana

Lithobates Phyllomedusa — Xenopus

Hylambates

Kassina Physalaemus Uperoleia Rana

Phylloseptins

Plasticins

Ranatuerins

Sauvagine Pancreatic polypeptide Spasmolysin

Tachykinins Hylambatin

Kassinin Physalaemin Uperolein Temporins

∗

Xenopus laevis

Bombina

Neurotensin

Thyrotropin-releasing hormone

Substance P, Substance K, Neuromedin K — — — Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ — —

Corticotropin-releasing factor Neuropeptide Y Breast cancer associated peptide (pS2) Pancreatic spasmolytic polypeptide

Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ — Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗ Cathelicidin LL-37/hBD1-3, RNase 7, psoriasin∗

Mammalian equivalent

Peptides with different structural properties to amphibian skin counterparts, despite an equivalent function at the same anatomical zone.

Thyrotropin-releasing hormone Xenopsin

Conlon (2008) Conlon (2008)

Phelophylax Lithobates

Palustrins

Hylarana Lithobates

Conlon et al. (2008a)

Odorrana

Zasloff (1987)

Xenopus

Magainins

Nigrocins

References

Representative genera

Peptide family

Table 2. Continued

Carraway & Leeman (1973, 1975)

Burgus et al. (1970)

Severini et al. (2002) and Kangawa et al. (1983) — — — Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) — —

Vale et al. (1981) Tatemoto et al. (1982) Nunez et al. (1987) Jorgensen et al. (1982)

Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) — Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b) Bernard & Gallo (2011), Glaser et al. (2005) and Zasloff (2009b)

References

Frog skin biology 637



638

(A)

(B)

(C)

(D)

Fig. 8. Schematic representation of the isolation and identification of frog skin antimicrobial peptides (AMPs). In frogs, AMPs are stored in granules of dermal serous glands (A). The skin secretion product can be obtained by giving the animal a mild electrical shock (10 V for 5 s) or a subcutaneous injection of noradrenaline (80 nmole per g body mass) (B), and then isolated by reverse-phase high-performance liquid chromatography (HPLC) (C). Peaks containing peptides with antibacterial activity (underlined region) can be identified by the inhibition zone assay (D): briefly, 3 mm wells are punched in 1% agar medium plates, containing approximately 400000 bacterial cells. The content of each HPLC fraction is dried and then resuspended in 20% ethanol. Three microlitres of each sample are loaded into the wells. After overnight incubation at 30◦ C, a transparent area (i.e. growth-inhibition zone) around the wells containing AMPs can be observed.

review of amphibian AMPs see Mangoni, 2009); a dedicated website (Wang, Li & Wang, 2009) contains information on all known AMPs. Importantly, each frog species produces its own unique set of AMPs, which constitute families of 100–200 closely related molecules (Mangoni, 2006). It is not possible to list all known AMPs here, but online Appendices S3 and S4 provide information on the principal classes from different anuran genera, i.e. Bombina, Rana, Xenopus and Phyllomedusa. These classes encompass bombinins (Mignogna et al., 1993), temporins (Simmaco et al., 1996), magainins (Zasloff, 1987) and dermaseptins (Mor, Amiche & Nicolas, 1994a; Mor & Nicolas, 1994), respectively.

VIII. FROG SKIN IMMUNOLOGY Whereas AMPs constitute the major player in the innate amphibian immune response, frog skin also displays adaptive immunity to pathogen infiltration, as shown by the identification of immunological components sharing features

with the mammalian skin immune system. Below we consider the main cellular components of this response: dendritic cells and mast cells.

(1) Dendritic cells Langerhans cells (LCs) in the mammalian epidermis, are dendritic, antigen-presenting cells that, once activated, interact with T-lymphocytes (Teunissen, 1992). It has been proposed that allograft rejection in amphibians results from antigen sensitisation, an idea leading Carrillo-Farga et al. (1990) to speculate on and discover the existence of LC homologues in frog skin. These cells were further characterised by Castell-Rodriguez et al. (1999), confirming the existence of ATPase-positive cells expressing major histocompatability complex (MHC) class II molecules. As such molecules are required for antigen presentation and subsequent T-cell recognition, frog and mammalian skin likely share molecular and cellular properties of adaptive immunity.


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639

A recent publication from Ramanayake et al. (2007) highlights the use of X. laevis as a comparative model for studying the immune response following skin grafting. Due to the ease of maintenance and breeding as well as its commercial availability, X. laevis has become an extensively studied organism in this field of immunology (Horton & Manning, 1972; Chardonnens & Du Pasquier, 1973; Flajnik & Kasahara, 2001). Allogeneic skin graft rejection in humans is mediated predominantly by influx of CD8+ T-lymphocytes, stimulated by the migration of LC from the grafted tissue into the recipient lymph nodes (Larsen et al., 1990). A similar mode of allograft rejection has now been identified in X. laevis (Rau, Cohen & Robert, 2001), providing an unique opportunity to present lessons learned from frog graft rejection research in the field of human allograft research. (2) Mast cells The involvement of mast cells in human innate and adaptive immunity has been extensively studied and particularly concerns leukocyte recruitment in inflammatory skin (Walsh et al., 1991), direct (chemotactic) or indirect (via induction of adhesion molecule expression in endothelial cells) stimulation

of T-cell migration (Mekori & Metcalfe, 1999), induction of LC migration following tumour necrosis factor α (TNFα) secretion (Jawdat et al., 2004; Suto et al., 2006) and eliciting a variety of responses by epidermal keratinocytes (production of pro-inflammatory cytokines, chemokines and growth factors) (Kohda et al., 2002). Numerous recent reviews describe human mast cell biology in some detail (Harvima, Nilsson & Naukkarinen, 2010; Kumar & Sharma, 2010; Molderings, 2010). By contrast, skin mast cell biology of non-mammalian vertebrates including frogs has not received a great deal of attention. Functionally, mast cells (Fig. 9) are believed to play similar roles in frogs and mammals, involving many identical molecular components (Baccari et al., 2011). Mast cell research in amphibians has largely focussed on the tongue and the nervous system (Baccari et al., 2011) and little work has been done to demonstrate the possible function(s) of these cells in frog skin. Mast cells in frog tissues have been described as displaying a close association with melanocytes, blood vessels and nerve fibre bundles (Baccari et al., 2011). Given the extraordinary immunological capacity of frog skin with its LC homologues and interactions with T-cells (Carrillo-Farga et al., 1990), as well as the production of glycosaminoglycans (indicated by metachromatic staining,

(A)

(C)

(B)

(D)

Fig. 9. Identification of mast cells in Xenopus laevis skin. Skin of X. laevis (zinc-fixation) embedded in paraffin and stained for mast cells (arrows in A & C). (A, B) Giemsa staining shows metachromasia of secretory granules (violet) in mast cells in the stratum spongiosum and stratum compactum. (C, D) Lede resterase staining reveals mast cells (red; pararosanilin) in the stratum compactum. Scale bars = 50 μm (A, C) and 20 μm (B, D). Biological Reviews 89 (2014) 618–655 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society


640 Fig. 9; Pelli et al., 2007), mast cell form and function in frog skin would likely prove to be a rewarding research topic.

IX. FROG SKIN PIGMENTATION (1) Chromatophore cell types Pigment-containing and light-reflecting cells in the integument of reptiles, amphibians and fish are known as chromatophores, and give rise to colouration of the skin and eyes of these poikilothermic species. All chromatophores arise from the neural crest, yet have distinct form and composition, with mature chromatophores characterised according to their hue under white light. In the dermis of most amphibians three types of chromatophore occur: xanthophores, iridophores and melanophores, arranged in dermal chromatophore units (Bagnara et al., 1968). The various chromatophores produce colours by either biochromes or schemochromes. The first contain true pigments such as carotenoids and pteridines, which selectively absorb part of the visible light spectrum making up white light, while permitting other wavelengths to reach the eye of the observer. Schemochromes, also known as ‘structural colours’, produce colouration by reflecting some wavelengths of light and transmitting others (Taylor, 1969; Morrison, 1995; Fujii, 2000). Xanthophores lie most superficially in the dermal chromatophore unit, directly under the epidermis. They contain pteridine pigments that give them a yellow, orange or red colour. If these colours are due to the presence of carotenoids, the cells are called erythrophores. Iridophores underlie xanthophores. They are silvery or white (in the latter case they are called guanophores) and reflect specific wavelengths of light through the overlying xanthophores. This reflection is caused by small platelets containing purine pigments that are arranged in stacks (Frost, Epp & Robinson, 1984). Generally, the iridophore layer is only one cell layer thick, but in species of the African frog, Chiromantis petersi (Rhacophoridae), 3–5 layers of iridophores occur. This demonstrates an important aspect of chromatophores in frog biology, namely their role in adaptation to environmental conditions, for which C. petersi is a good example (Drewes et al., 1977) (see Section V). The dermal chromatophore unit in C. petersi, consisting of several layers of iridophores, increases blue light reflectance, making the animal’s skin look red (Duellman & Trueb, 1994). Whereas some melanophores show an epidermal localisation (Duellman & Trueb, 1994), most are located basally in the dermal chromatophore unit. Melanophores are large cells with numerous, finger-like cytoplasmic processes that extend upward and end on the surface of iridophores. Their main pigment is eumelanin, which absorbs most of the visible light, resulting in a black-brown cell appearance (Bagnara et al., 1979; Ito & Wakamatsu, 2003). The pigment-containing melanosomes may be concentrated in the centre of the cell around the nucleus

(skin looks white) or widely scattered across all cytoplasmic processes, giving the skin a dark appearance. Melanophores and their melanosomes form an excellent model for the study of molecular mechanisms that coordinate intracellular organelle transport. Melanosome transport is reversible and involves both actin (myosin V) and microtubule-dependent (kinesin II and dynein) proteins (Sugden et al., 2004). The way light and various inhibitory and stimulatory hormones control the frog dermal chromatophore unit appears to be highly complex (see e.g. Bagnara et al., 1968). Most is known about the control of melanophores, which is influenced by a large variety of hormonal stimuli including α-MSH, noradrenaline, vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide-β (CGRP-β), melatonin, adrenaline, acetylcholine, histamine, serotonin and endothelin (see e.g., Sugden et al., 2004; Salim & Ali, 2011). Below we discuss the two most widely investigated hormones in this respect, α-MSH and melatonin. (2) α-MSH It has long been known that α-MSH, a 13 amino acid residue neuropeptide derived from the protein precursor molecule pro-opiomelanocortin (POMC), is responsible for neuroendocrine control of melanin distribution in frog skin during the animal’s adaptation (camouflage behaviour) to ambient light intensity (Bagnara et al., 1973). This process is very pronounced in X. laevis, where it has been studied extensively (e.g. Roubos et al., 2010a,b; Jenks et al., 2011). On a black background, α-MSH is released from neuroendocrine melanotrope cells in the pituitary pars intermedia (Fig. 7) to stimulate the dispersion of the melanin-containing pigment granules in dermal melanocytes (or ‘melanophores’) so that the animal achieves a darker appearance. On a white background α-MSH release is inhibited and as a result the pigment granules spontaneously aggregate around the cell’s nucleus, turning the skin pale. Research on R. esculenta and X. laevis has identified a plethora of classical neurotransmitters and neuropeptides that control the release of α-MSH (Fig. 7) (e.g. Tonon et al., 1993; van Wijk, Meijer & Roubos, 2010; Roubos et al., 2010a,b; Jenks et al., 2011). These messengers are produced in various parts of the brain and released from the neural pituitary lobe towards the melanotrope cells or reach them directly via synaptic contacts, permitting the animal to adjust skin pigmentation not only to ambient light condition, by γ -aminobutyric acid (GABA), dopamine and NPY (Tonon et al., 1983, 1989; Adjeroud et al., 1986; de Rijk, van Strien & Roubos, 1992; Tuinhof et al., 1993) but also in response to stressful situations, and following changes in ambient temperature and subsequent signalling by TRH (Tonosaki et al., 2004). The latter potent amphibian secretagogue for α-MSH (Tonon et al., 1980, 1983; Lamacz et al., 1987) is not only involved in mammalian temperature control (Mazzoccoli et al., 2004) but has also recently been found to stimulate melanophore pigment production/melanocyte activity in human hair follicles (Gaspar et al., 2011).


Frog skin biology

641

Interestingly, an increased α-MSH titre seems to cause skin darkening in humans too, especially in pregnant women where it is concomitant with an increased estrogen titre (Altmeyer et al., 1989). Moreover, in red-haired individuals, and those who do not tan well, the numbers of melanocortin receptors in the skin may be low, making them particularly sensitive to α-MSH (Smith et al., 1998). In comparison to the extensive knowledge of the control of α-MSH release from the frog pituitary, the control of α-MSH in the mammalian, including human, pituitary gland is poorly understood, although dopamine, the most potent inhibitory messenger found in frogs, may also control human pituitary α-MSH release (Pivonello et al., 2007). This makes the frog pars intermedia and its α-MSH control system an interesting model for studying α-MSH release and its physiology in man. (3) Melatonin Melatonin is synthesized and released from the pineal gland mainly at night, and regulates various aspects of physiology and behaviour, including circadian and seasonal responses, and some retinal, cardiovascular and immunological functions. In amphibians such as X. laevis tadpoles, another role of melatonin is in the control of skin colouration through action on the melanin-containing pigment granules (melanosomes) in melanophores. The hormone is thought to act only before metamorphosis; after this process, α-MSH takes over melanophore control in relation to background light intensity (Sugden et al., 2004). The role of ocular melatonin has been investigated (Wright et al., 2006) by ophthalectomy in Rana catesbeiana tadpoles and froglets, with results indicating the significant involvement of the eye in the release of melatonin into the circulation. The reduced plasma melatonin titre in these animals also corresponds to general skin darkening, independent of changes in α-MSH concentration, providing further evidence for the role of melatonin in skin lightening (Wright et al., 2006). Indeed, the skin-lightening effect of a pineal extract that was termed ‘melatonin’ was first observed over 50 years ago by Lerner et al. (1958). Skin colour change in frogs appears to be species dependent, with one common feature being the ability of α-MSH to induce skin darkening, as described above (Camargo, Visconti & Castrucci, 1999). Melatonin, on the other hand, does not always display consistent effects, with recognised antagonism of α-MSH in R. pipiens and X. laevis, whereas in R. catesbeiana only partial skin lightening has been observed (Camargo et al., 1999). (4) Comparison with mammalian chromatophores The presence of an integrated pigmentary unit in frog skin, containing the structural and functional arrangement of distinct cell types (xanthophores, iridophores and melanophores/melanocytes) in a dermal chromatophore unit, explains how frog skin is able rapidly to alter skin colour. By contrast, the mammalian epidermal chromatophore unit functions quite differently: xanthophores and iridophores are absent and each unit comprises a melanocyte and its

associated keratinocytes, in which melanin is distributed along dendritic cell extensions into the epithelial acceptors that produce the characteristic skin darkening stimulated by UV irradiation. Unlike frogs, human skin contains only melanocytes and fully differentiated, pigment-producing melanocytes are restricted to defined intraepithelial compartments, specifically the basal layer of the epidermis and the epithelial hair bulb (Slominski et al., 2005). The pigments within human melanophores are a combination of both eumelanin (black-brown) and pheomelanin (redbrown), whereas amphibian skin contains only the eumelanin form (Bagnara et al., 1979; Ito & Wakamatsu, 2003). Frog skin darkening is easily reversible, with resequestration of the melanin-containing granules exposing the underlying iridophore and xanthophore cells of the chromatophore unit, returning a lighter colouration (Bagnara et al., 1968). This rapid colour change in Anura contrasts sharply with changes brought about by the mammalian epidermal melanin unit, where melanin is contained in granules that cannot move inside the cytoplasm but are physically released from the melanocyte dendrites towards the surrounding keratinocytes. Similarly, pigmentation reduction in mammalian skin is a much slower process than in frogs, and cannot occur until the released melanin has been degraded or dispersed (Bagnara et al., 1968).

X. TOOLS FOR FROG SKIN RESEARCH Unlike some of their mammalian counterparts, the genomes of amphibians are generally not well characterised, and most commercially available antisera are not designed for use in species so evolutionarily distant from mammals. As such it can often seem difficult to probe the molecular basis of frog skin structure and function. Nevertheless, besides classical histochemistry, a number of advanced techniques are available that facilitate research into the anatomy, physiology and biochemistry of frog skin (see Table 3). Frog skin organ culture methods have existed for decades (Uhlenhuth, 1914) and have provided invaluable insights into epithelial migration, proliferation, barrier function and transport capabilities. Traditional histochemical techniques, immunohistochemistry and (immuno-)electron microscopy of fresh and cultured frog skin have provided much of our knowledge regarding the fine structure of the amphibian integument (e.g. Voute, 1963; Yoshii et al., 2005a,b). The body of work produced by Denefle & Lechaire (1990, 1992) has amply demonstrated the usefulness of optimised frog skin organ cultures in the study of pigment cell behaviour, wound healing (Zhu, Denefle & Lechaire, 1986) and epithelial cell migration and differentiation (Denefle & Lechaire, 1984; Denefle, Lechaire & Zhu, 1987). These methods have recently been complemented by a serum-free frog skin organ culture technique that facilitates comparative analysis with human skin (Meier et al., 2013). In conjunction with traditional morphological analyses of frog skin, the application of new antisera in



642 Table 3. Available tools for frog skin research Technique

Research topic

References

Skin organ culture

Long-term study of amphibian skin in culture

Monnickendam & Balls (1973), Yoshii et al. (2005a,b),

Wound repair model

Electrophysiology (Ussing chamber) Electron microscopy

Ion and solute transport Osmoregulation Study of integument/fine study of epidermis Study of wound repair

Mass spectrometry Histology and immunohistochemistry

Transgenic frogs

Characterisation of integumental peptides Study of integument/epidermis Location of structural proteins Study of integument glands Cellular immunity

Regenerative capacity of frogs Mechanisms of wound repair in the integument

immunohistochemistry, the availability of specific DNA probes for in situ hybridization and the use of proteomics and peptidomics (e.g. King, Neff & Mescher, 2009) will allow a greater understanding of its structure, immunocompetence and regenerative capacity. Transgenic technology in particular has become an important research tool in regenerative medicine and has also been utilised in developmental and regenerative studies in X. laevis (e.g. Beck, Izpisua Belmonte & Christen, 2009). In elucidating the primary structure of the extraordinary array of active peptides secreted by the skin of numerous amphibian species, mass spectrometry has proven to be an invaluable resource; matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry combined with rapid sequencing of isolated fractions has been shown to be an efficient approach (Conlon et al., 2007a; Thompson et al., 2007). Taken together, these multidisciplinary research tools invite instructive comparative analyses of adult frog, murine and human skin in vitro, ex vivo and in vivo.

XI. TRANSLATIONAL ASPECTS In the previous sections of this review, it has been demonstrated that frog skin is endowed with numerous features that are similar to the human integument, but also displays many aspects that differ considerably. From a scientific point of view, it is both interesting and rewarding to

Meier et al. (2013), Denefle et al. (1989), Denefle & LeChaire (1984), Derby (1978) and Uhlenhuth (1914) Ussing & Zerahn (1951) Weiss & Ferris (1954) Voute (1963) Parakkal & Matoltsy (1964), Felsemburgh et al. (2009) Yoshii et al. (2005a,b) Thompson et al. (2007) Parakkal & Matoltsy (1964) Zhu (1987) Denefle et al. (1993) Vanable & Mortensen (1966) Bagnara et al. (1968) Mescher et al. (2007) Beck et al. (2009) Pearl et al. (2008) Robert & Ohta (2009)

highlight these parallels and differences in skin structure and function, as they result from the gradual process of evolution and distinct environmental pressures of adaptation and selection. (1) Barrier function The expression of tight junctional protein complexes forms a physical barrier in the skin of frogs and humans, albeit a much leakier version in Anura (Martinez-Palomo et al., 1971; Shahin & Blankemeyer, 1989; O’Neill & Garrod, 2011). Whereas tight junctions in frog skin have previously been studied in relation to the ion-transporting function of mammalian epithelium such as the intestine and kidney tubules, little attention has been given to their role in the skin. Indeed, although in the early work of Farquhar & Palade (1965) and Martinez-Palomo et al. (1971) anuran integumental junctional complexes and permeability barriers were localized and characterised (stratum corneum and stratum granulosum), subsequent research investigating the molecular composition of these structures is sadly lacking. By contrast, the fundamentals of junctional protein expression in the mammalian epidermis, including, for example, the relative importance of claudin 1 and Ecadherin proteins in transepidermal permeability barrier homeostasis are well established (O’Neill & Garrod, 2011). In mammalian skin, barrier function was traditionally believed to result from interplay between the lipid lamellae and the highly cross-linked cornified envelope, producing an effective and insoluble cutaneous permeability barrier. More


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recently, the importance of tight junctional complexes in the maintenance of the skin barrier has been discussed (O’Neill & Garrod, 2011). Deletion of claudin 1 in mice was found to result in severe dehydration and neonatal death through massive transepidermal water loss, yet the lipid lamellae and cornified envelope were unaltered (Furuse et al., 2002). Whilst the barrier properties of frog skin are significantly different to those of human skin (particularly with respect to overall gas and fluid permeability), frog skin may still represent an appealing model with which to study tight junctional functions, such as selective permeability, and the relative contribution of individual tight junctional proteins to this process. Another intriguing aspect of tight junctions is their recently identified role in the immune response (Shaw & Huang, 2010). In addition to providing a physical barrier to the invasion of external pathogens, junctional complexes are now recognised to interact with dendritic epidermal T cells (DETCs). It has been hypothesised that disruption of tight junctions by, for instance, viral infection will cause activation of DETCs via putative interactions of coxsackievirus and adenovirus receptor, sequestered within junctional complexes, and junctional adhesion moleculelike protein, expressed on the DETCs (Shaw & Huang, 2010). Interactions between anuran epidermal dendritic cells and junctional complexes remain to be identified, but the pathogen-rich environments in which frogs are generally located would make a similar immunosensory function extremely beneficial. (2) Anti-infection defence The role of AMPs in host-defence mechanisms against microbial infections is manifested by the fact that their functional failure drives the emergence of different immunocompromised phenotypes, both in animals and humans (Putsep et al., 2002). The widespread use of antibiotics has drastically reduced their therapeutic efficacy due to the growing appearance of multidrug-resistant bacterial strains. As this has serious health implications, especially in hospitals, the development of novel antiinfection strategies with alternative mechanisms of action is urgently needed. In this context, AMPs have attracted the interest of the clinical community. Note that pexiganan (a derivative of the frog skin AMP magainin) failed to pass the Food and Drug Administration (FDA) approval for the treatment of infected diabetic foot ulcers, not because of its inefficacy to cure infection, but rather due to its similar activity to oral ofloxacin (Lipsky, Holroyd & Zasloff, 2008). However, no significant bacterial resistance to pexiganan emerged among patients enrolled in a large-scale clinical trial involving more than 800 individuals (Lipsky et al., 2008). This is in contrast with the bacterial resistance to ofloxacin found in patients receiving this antibiotic. Importantly, we should not generalize the outcome of pexiganan to all AMPs and derivatives from frog skin, as the success of a peptide to be used for therapeutics is highly related to its particular biophysical properties (sequence, hydrophobicity, oligomeric

state etc.), tissue penetrability and cell specificity. There are clear precedents for natural peptides having clinical efficacy, such as polymixin used to treat Pseudomonas and Acinetobacter infections and the lipopeptide daptomycin used to treat skin infections by Gram-positive bacteria (Fjell et al., 2012). Many studies are currently directed at optimising the properties of native AMPs (their length and amino acid residue sequence, while improving their resistance to proteases, stability and half-life time) to formulate next-generation peptide-based antimicrobial drugs (Shai & Oren, 1996; Hancock & Sahl, 2006; Mangoni & Shai, 2011). Although compared to frog skin, human skin does not possess nearly as diverse a complement of AMPs (Bevins & Zasloff, 1990; Schittek et al., 2008; Wiesner & Vilcinskas, 2010), particular AMPs may become important tools in human pathogen protection (Rieg et al., 2004; Reithmayer et al., 2009; Zasloff, 2009a,b). The major AMPs in human epidermis are synthesized by keratinocytes in the stratum granulosum, and are delivered into the stratum corneum, specifically to the lamellar bodies therein (Braff, Di Nardo & Gallo, 2005; Aberg et al., 2007), where they contribute to the formation of a barrier to water loss and microbial attack (Aberg et al., 2008). Sunlight induces keratinocytes to produce the cathelicidin AMP, LL-37, via a vitamin D-mediated circuit, thus explaining the efficacy of phototherapy in the treatment of cutaneous tuberculosis (Mallbris et al., 2005; Zasloff, 2006). Importantly, besides providing an antimicrobial skin barrier, LL-37 could limit the extent of skin burns through its antiapoptotic effect (Chamorro et al., 2009; Zasloff, 2009a). Rieg et al. (2004) identified the constitutive expression of dermcidin in human sweat glands, from where it is secreted onto the skin surface along with other components of sweat, thus preventing local and systemic invasion by microorganisms rather than responding to chemical signals associated with injury and inflammation. The presence of AMPs has also been confirmed in the human hair follicle and its associated apocrine gland, including human βdefensins (hBD1 and hBD2), as well as the S100 protein, psoriasin and RNase 7 (Reithmayer et al., 2009; Zasloff, 2009b). Recently, upregulated expression of hBD1, 2 and 3 as well as of psoriasin has been reported for human oral mucosa but not in the extraoral skin (Kesting et al., 2011). Given the similarities in functionality and expression in skin appendages, comparative AMP research on frog and human skin may increase our understanding of skin defence mechanisms against pathogens. Such an approach would also seem interesting in view of the comparatively moist nature of frog skin, which may more closely resemble the human oral mucosa than human truncal skin. A number of human skin disorders are associated with disrupted barrier function and dysregulation of the immune response. One common pruritic inflammatory skin disease, atopic dermatitis (AD), is characterised by patches of dry, flaky skin that are prone to recurrent infection. In addition to findings associating the disease state to defects in the



644 epidermal permeability barrier (Palmer et al., 2006) other findings link this condition to an altered expression of AMPs (Ong et al., 2002). As recent evidence has shown that the permeability barrier and antimicrobial defences are inextricably linked, stressors that impact on skin barrier functions are also likely to impact on AMP secretion (Elias & Choi, 2005; Aberg et al., 2008). One such example is psychological stress, which has been shown to both impair murine skin barrier function and reduce AMP expression in a glucorticoid- and β-adrenergic-dependent manner (Aberg et al., 2007; Martin-Ezquerra et al., 2011). Development of new treatments based on targeting specific pathways that modulate AMP production (Antal et al., 2011) could be promising, given that current practice in the treatment of AD by topical application of corticosteroids reduces the skin concentration of AMPs, thereby increasing the risk of infection without restoring the skin physical barrier (Jensen et al., 2011). This is likely the result of impaired epidermal lipid biogenesis, which would impact on both lamellar body formation and also, therefore, on skin permeability and AMP secretion (Kim et al., 2010; MartinEzquerra et al., 2011). Inhibition of de novo biosynthesis of frog skin AMPs by glucocorticoid treatment [presumably due to increased production of the nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) inhibitor protein, IκBα] was demonstrated in R. esculenta (Simmaco et al., 1997, 1998; Mangoni et al., 2001). Should a treatment become available that can maintain normal AMP levels while also providing the clinical benefits associated with corticosteroid treatment (antiinflammatory effects, restoration of disturbed differentiation), long-term AD therapy without atrophic side-effects will be feasible. (3) Immunology With the recent publication of the X. tropicalis genome (Hellsten et al., 2010), complementing the X. laevis mitochondrial genome publication of some years ago (Roe et al., 1985), a host of new tools and techniques have opened up for immunologists interested in frog-mammalian comparative research (Robert & Ohta, 2009). For some years X. laevis has been the chosen experimental frog, linking mammalian immunological studies to evolutionarily more ancient vertebrate species, in particular possessing an intriguingly similar adaptive immune system (Robert & Ohta, 2009). A review by Robert & Ohta (2009) describes in great detail the conserved and novel features of X. laevis, X. tropicalis and mammalian immune systems. Of particular importance to mammalian skin researchers with an interest in the immunological functions of the integument, are the inbred homozygous MHC strains of X. laevis, which allow the classical genetic manipulations so common in modern murine research (Robert et al., 2004). It is envisaged that similar strains will soon be available for X. tropicalis, providing a novel source of experimental animals for immunological research (www. urmc.rochester.edu/mbi/resources/Xenopus/). In this way

valuable insights may be gained into the evolutionarily conserved features of vertebrate immune responses in many organs including skin. The number of frog species is in global decline, with extinction threats coming from various sources, not only from environmental disruption/destruction, predation and pollution, but also from fungal (e.g. Batrachochytium dendrobatidis) and viral pathogens (e.g. Chen & Robert, 2011). Understanding the susceptibility of frogs to these pathogens is a major challenge (Chen & Robert, 2011). Possibly, comparative research into the immunological responses to viral pathogens in distinct frog species could yield useful information to immunologists primarily studying mammals. Frogs are already used to study adaptive immune responses, using antisera raised against pathogens (Ramsey et al., 2010), antigen-specific skin transplant rejection systems (Arnall & Horton, 1986; Tozaki & Tochinai, 1998) and mammalian Langerhans cell homologues involved in T-cell recruitment (Castell-Rodriguez et al., 1999; Rau et al., 2001). Together with advanced antiviral skin studies, these approaches may provide excellent tools for the comparative skin immunobiologist. (4) Aquaporins AQPs have already been discussed in relation to water uptake (Section V.3). Our understanding of AQP function in mammalian skin is still in its infancy, with conflicting reports as to the relevance of AQP activity in maintaining normal skin function. Yet the hormonal control of the expression and activity of AQPs in frog skin is well established, unlike the situation for human skin. Of particular interest to human skin researchers is AQP3, which plays a role in various aspects of frog skin health (Ishibashi et al., 2011). It is likely that AQP3 transports glycerol, since topical glycerol application reverses skin dehydration in AQP3-null mice (Verkman, 2009; Ishibashi et al., 2011), and it also stimulates keratinocyte proliferation as its overexpression promotes the development of basal cell carcinoma. Therefore, AQP3 inhibitors may assist in treating human skin cancer (Verkman, 2009; Ishibashi et al., 2011). In addition, upregulation of AQP3 may be involved in the epidermal hyperplasia associated with the onset of AD (Nakahigashi et al., 2011), whereas its downregulation may facilitate the development of vitiligo (Kim & Lee, 2010). Gene expression profiling has also shown that AQP dysregulation occurs in psoriasis, with AQP5 and AQP9 deficiencies having been identified (Suarez-Farinas et al., 2011). Consequently, although the involvement of AQPs in human skin health and disease is increasingly recognised, their various roles in normal physiological skin function and the impact thereon of their disruption is not well understood and deserves attention. (5) Sebaceous and mucus glands Human dermis houses both sebaceous and sweat glands, with the exocrine sweat gland in particular playing a vital role in thermoregulation. The sebaceous gland secretes oily sebum to lubricate the skin and has also been implicated in the


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development of acne (Kligman, 1963; Downing et al., 1987). Anuran mucus glands are functionally analogous to human sweat glands as they have a thermoregulatory function, but are structurally quite distinct. In both human sweat glands and amphibian mucus glands, the composition of secretions is controlled by the ionic transport function of the enclosing epithelia, with the electroneutral co-transport of Na+ , K+ and Cl- (1:1:2) being the predominant mechanism to maintain osmotic balance and, hence, proper water movements (Sato et al., 1989; Ussing et al., 1996). The fluid secreted by amphibian mucus glands is generally isotonic (Ussing et al., 1996) and although human sweat is hypotonic compared to plasma, it is formed from an isotonic precursor fluid (Mangos, 1973). Therefore, frog mucus glands offer excellent models for studying the formation of isotonic secretions by mammalian skin (Ussing et al., 1996). The anuran granular gland is of great interest to human skin researchers because of its role in defence against microbial infection and predators. Studying the compositions and functions of its secretions is very interesting in terms of chemical prospecting of new lead compounds in pharmaceutical development (Clarke, 1997; Conlon et al., 2008b; Raghavan et al., 2010). Other applications of these secretions can also be imagined, such as in the areas of biomaterials and bioengineering. For instance, sticky dermal substances secreted by some frog species (Evans & Brodie, 1994) could be used as adhesives to aid in the closure of human skin wounds (Clarke, 1997). The use of ‘bioglues’ has been extensively investigated since the review of Clarke (1997), particularly in the fields of cardiac and respiratory surgery (Belcher et al., 2010; Lawrence, Shah & Yang, 2011), but thus far compounds developed from frog skin secretions have not found clinical application. (6) Tissue regeneration/wound healing Frog skin shows highly efficient wound repair, particularly in larval anurans during complete appendage regeneration (Harty et al., 2003; Yokoyama et al., 2011). The complex mechanisms of wound healing in the skin are, to a degree, conserved between frogs and humans, and generally consist of three primary phases: (i) inflammation, (ii) migration and proliferation, and (iii) remodelling. Wound healing in adult frog skin is dependent upon developmental stage, with progression of morphogenesis coupled to a decline in regenerative capacity (Suzuki et al., 2006). However, a recent study of young adult X. laevis froglets (Yokoyama et al., 2011) demonstrated scarless healing akin to foetal wound healing in mammals (Ferguson & O’Kane, 2004). The efficient healing described in X. laevis froglets has been attributed in part to the paired-type homeobox gene pxr1, which appears to be absent in adult mouse skin wounds (Yokoyama et al., 2011). As such, a loss of pxr1 expression in adult mammals could present a possible mechanism for their relatively poor healing ability compared to frogs. Investigating such signalling mechanisms in frog species such as X. laevis could therefore represent an attractive model for studying scarless wound repair.

Another potential avenue that warrants investigation follows increasing evidence that AMPs could play a role in wound healing. Upregulation of the AMP cathelicidin by growth factors released upon cutaneous wounding is believed to play a role in preventing infection upon disruption of the skin barrier (Sorensen et al., 2003). Cathelicidin may also promote re-epithelialisation of wounds and, through increased angiogenesis, in improving vascularisation (Heilborn et al., 2003; Ramos et al., 2011). An important finding by Heilborn et al. (2003) is that chronic non-healing ulcers lack cathelicidin expression and that inhibition of cathelicidin prevents re-epithelialisation in a human in vitro organ culture wound-healing model.

XII. CONCLUSIONS (1) Having described frog skin biology in detail, differences between frog and mammalian skin biology have been highlighted. (2) Frog skin environmental adaptations are extraordinarily diverse, having developed to meet a range of challenging habitats and notably encompassing both aquatic and terrestrial living. Whilst many specific frog skin features represent unique solutions to specific environmental challenges, there are particular anatomical structures, biologically active molecules and processes that are conserved in mammalian skin. (3) The study of frog skin offers many fascinating insights into several areas of skin research that may be useful for biologists and investigative dermatologists alike. (4) While frog skin is worthy of investigation in its own right from a conservation and ecological perspective (particularly given the challenges faced by the threat of chytridiomycosis), we expect that mammalian skin researchers will increasingly use frogs to enhance their knowledge of mammalian, and especially human, skin structure and function.

XIII. ACKNOWLEDGEMENTS The authors wish to acknowledge Professor Enrique Amaya and Dr Roberto Paredes for their most helpful review and comments on this manuscript. The authors acknowledge with thanks the use of many images from Internet sources in the preparation of Figs 1 and 2: specific attributions are given in the form of reference citations in the relevant figure legends.

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XV. SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article. Appendix S1. Frog skin design in an evolutionary context and comments on their piscine ancestors. Appendix S2. Principles of frog skin development. Appendix S3. Principal classes of antimicrobial peptides (AMPs). Appendix S4. The pharmacologically active peptide bombesin.

(Received 3 February 2012; revised 3 October 2013; accepted 22 October 2013; published online 3 December 2013)


Peptides from frog skin.

Bombesin-like peptides: from frog skin to human lung.

Antimicrobial peptides of frog skin.

Wash out characteristics of tracer Na from the transport pool of frog skin.

Deviations from the flux ratio equation for chloride ions in ouabain- and acetazolamide-treated frog skin.

Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa.

A Novel Insulinotropic Peptide from the Skin Secretions of Amolops loloensis Frog.

Magainin 2, a natural antibiotic from frog skin, forms ion channels in lipid bilayer membranes.

Electrophysiological effects of a neurotoxin extracted from the skin of the Australian frog Pseudophryne coriacea.

Identification and characterization of two dermorphins from skin extracts of the Amazonian frog Phyllomedusa bicolor.

Six novel tachykinin- and bombesin-related peptides from the skin of the Australian frog Pseudophryne güntheri.

A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

Paxillin and steroid signaling: from frog to human.

Pharmacology of 5-hydroxytryptamine receptors in frog skin epithelium.

Response characteristics of a multicompartment frog skin epidermis model.

Localization of pigment cells in cultured frog skin.

Network thermodynamic approach compartmental analysis. Na+ transients in frog skin.

Deviating flux ratios for Na+ in ouabain-treated frog skin.

Inhibition of potassium conductance by barium in frog skin epithelium.

The mechanism of action of Cu2+ on the frog skin.

Transient current changes and Na compartimentalization in frog skin epithelium.

Regulation of ion conductance in frog skin by isoproterenol.

Endorphins supersensitize frog skin melanophores to isoproterenol, but subsensitize them to alpha-melanocyte-stimulating hormone.

Computer modelling. Application to studies on the initial rate of Na+ uptake by frog skin epidermis.