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The Journal of Experimental Biology 216, 2478-2486 © 2013. Published by The Company of Biologists Ltd doi:10.1242/jeb.082396

REVIEW Electric : new insights into conserved processes of adult tissue regeneration

Graciela A. Unguez Department of Biology, New Mexico State University, Las Cruces, NM 88003, USA [email protected]

Summary Biology is replete with examples of regeneration, the process that allows to replace or repair cells, tissues and organs. As on land, vertebrates in aquatic environments experience the occurrence of injury with varying frequency and to different degrees. Studies demonstrate that ray-finned possess a very high capacity to regenerate different tissues and organs when they are adults. Among fishes that exhibit robust regenerative capacities are the neotropical electric fishes of (Teleostei: ). Specifically, adult gymnotiform electric fishes can regenerate injured brain and spinal cord tissues and restore amputated body parts repeatedly. We have begun to identify some aspects of the cellular and molecular mechanisms of tail regeneration in the weakly macrurus (long-tailed knifefish) with a focus on regeneration of and the muscle-derived . Application of in vivo microinjection techniques and generation of myogenic stem cell markers are beginning to overcome some of the challenges owing to the limitations of working with non-genetic models with extensive regenerative capacity. This review highlights some aspects of tail regeneration in S. macrurus and discusses the advantages of using gymnotiform electric fishes to investigate the cellular and molecular mechanisms that produce new cells during regeneration in adult vertebrates. Key words: epimorphic regeneration, electric fish, satellite cells, muscle regeneration, tail regeneration, electric organ, Sternopygus, dedifferentiation. Received 30 October 2012; Accepted 13 December 2012

Introduction ‘tadpoles’ easily regenerate their limbs (Muneoka et al., 1986). Biology is replete with examples of regeneration, the process that Progressive decline in the regenerative response to limb amputation allows animals to replace or repair cells, tissues and organs. with increasing age has also been demonstrated in rodents Regeneration provides obvious benefits to survival and function (Deuchar, 1976; Lee and Chan, 1991; Wanek et al., 1989). In and is known to occur widely among animal phyla (Bely, 2010; mammals, skeletal muscle is considered a highly regenerative Sánchez Alvarado and Tsonis, 2006). Some animals perform this tissue as it can repair itself after injury or disease. Similar to limb remarkable feat almost flawlessly. However, the ability to fully regeneration, aging skeletal muscle shows a diminished ability to regenerate complex tissues decreases as one moves from the basal repair itself and an increased susceptibility to contraction-induced groups in the animal kingdom to those more highly derived groups injury (Alnaqeeb and Goldspink, 1987; Brooks and Faulkner, (Goss, 1969). For example, invertebrates including jellyfish, 1994). Failure to repair a damaged region of muscle fiber can result planarians, segmented worms, mollusks and starfish can fully in severe cell atrophy and even cell death. This is in stark contrast regenerate multiple tissues and even entire body parts (Brusca and to the inexhaustible regeneration of skeletal muscle tissues and Brusca, 1990; Ferretti and Geraudie, 1998; Brockes and Kumar, even entire body parts exhibited by some adult non-mammalian 2002). Among vertebrates, amphibians and fishes generally express species (Unguez and Zakon, 1998; Kirschbaum and Meunier, 1988; a robust regeneration response to limb loss, whereas reptiles, birds Sánchez Alvarado and Tsonis, 2006). and mammals have a more limited capacity to regenerate complex Regeneration processes are grouped according to the presence or body structures (Brockes and Kumar, 2008; Galis et al., 2003). The absence of cell division during the replacement of lost tissues evolutionary loss of regenerative ability in some groups, to varying (Morgan, 1901). Morphallaxis occurs in the absence of cell degrees, remains an unsolved problem that attracts many biologists proliferation, and epimorphosis requires cell proliferation. (Bely and Nyberg, 2010). And, not surprisingly, the limited Epimorphosis is further categorized according to whether a capacity of humans to regenerate and restore tissues and organs regeneration blastema forms. A blastema is a specialized fuels enormous interest from a medical perspective in elucidating proliferative zone of mesenchymal cells that forms beneath the the mechanisms responsible for the extensive regenerative epidermis at the wound site. Blastema formation is a key process capacities in vertebrates. in epimorphic regeneration in both invertebrates and vertebrates In general, studies exploring regeneration show that vertebrate (Thouveny and Tassava, 1998). The widespread distribution of this species that can restore amputated limb structures during early feature across animal taxa supports the hypothesis that a program development exhibit little to no regenerative capacity as adults. For for epimorphic regeneration may have been conserved through example, all adult frogs either do not regenerate their limbs at all (Brusca and Brusca, 1990; Sanchez-Alvarado, 2000). To or they regenerate a spike of cartilage surrounded by skin – an date, the general notion is that a blastema is formed primarily from ‘outgrowth’, but hardly a limb – whereas all immature frogs or the re-entry of mature cells near the injury site into the cell cycle,

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Fig.1. Tail amputation in an adult zebrafish Danio rerio. (A)An adult control fish. Dotted line shows site of tail amputation immediately proximal to anal fin. (B)The same zebrafish 2weeks after amputation. Note the blastema formed at the base of the amputation plane that appears as a visible swelling at the end of the tail. Photo credit: Michael McDowell. i.e. dedifferentiation, to contribute to the restoration of lost tissue. Regeneration in adult zebrafish In fact, the idea that a greater potential for epimorphic regeneration Research in fish regeneration biology has focused largely on the is associated with a corresponding potential for cell zebrafish, as it possesses a large regenerative capacity and is an dedifferentiation remains broadly accepted (Brockes and Kumar, ideal model system for investigation using cellular, molecular and 2002; Nye et al., 2003; Tsonis, 1991). Support for this idea is based genetic approaches and (Poss et al., 2003; Akimenko et al., 2003). largely on the vast body of evidence from experiments in urodele Studies on zebrafish regeneration are contributing extensively to amphibians such as the newt and axolotls, which are able to our current understanding of regeneration mechanisms to replace regenerate limbs, tail, jaws, ocular tissues including retina and lens, different cell and tissue types. Indeed, the ability to carry out loss- and some portions of the heart. Specifically, in vivo cell tracing and gain-of-function experiments in combination with genetic experiments in urodele amphibians have shown that mature cells screens and in vivo cell tracing is increasing the identification of including muscle fibers can dedifferentiate, proliferate and enter the regulatory factors and progenitor cells involved in zebrafish blastema (Stocum, 1995; Echeverri et al., 2001; Brockes and regeneration (Tanaka and Reddien, 2011; Sánchez Alvarado and Kumar, 2002; Nye et al., 2003; Nechiporuk and Keating, 2002; Tsonis, 2006; Singh et al., 2012). Even so, the ability of adult Tanaka and Reddien, 2011). Similarly, the robust regenerative zebrafish to fully repair and restore all damaged or lost tissues, capacity of zebrafish to regrow amputated fins, heart and neural organs and limbs is limited. For example, a zebrafish cannot tissue such as the spinal cord and retina is suggested to depend on regenerate its tail if amputation occurs proximal to the caudal fin dedifferentiation of cells near the amputation site by studies using (Fig.1). In contrast, many South American gymnotiform electric transgenesis, loss- and gain-of-function technology, and cell fish species can repeatedly regenerate their tails following labeling methodology (Tanaka and Reddien, 2011; Singh et al., amputation during adulthood. 2012; Poss et al., 2003; Poss, 2010). Although cell dedifferentiation might be favored under certain Regeneration in adult gymnotiform electric fish conditions, it is not possible to exclude the contribution of There are more than 700 vertebrate species known that are capable alternative modes of regeneration. For example, generation of of generating and all of them are fishes representing 11 reliable cell markers have led to recent findings that resident, tissue- independent lineages (Albert and Crampton, 2006). Of these, specific stem cells also respond to injury and contribute to blastema approximately 173 species are gymnotiforms, or South American formation in some vertebrate species (Li et al., 2007; Morrison et knifefishes, and in all but one group, the electric organ derives from al., 2006; Poss et al., 2003; Singh et al., 2012; Weber et al., 2012). striated muscle cells that suppress many muscle phenotypic Hence, the extent to which cell dedifferentiation versus stem cell properties (Bennett, 1971). Gymnotiforms have been used to study activation occurs in response to injury in vertebrates warrants the formation of sensory and motor specializations (Fox and further investigation. Interestingly, multiple regeneration responses Richardson, 1978; Fox and Richardson, 1979; Kirschbaum and may come into play following injury or disease in the same animal. Schwassmann, 2008; Denizot et al., 1998; Denizot et al., 2007; In mammals, as in zebrafish, liver regeneration is accomplished Vischer, 1989a; Vischer, 1989b; Zakon, 1984; Lannoo et al., 1990), largely by hepatocyte dedifferentiation (Kan et al., 2009; Fausto et (Zakon et al., 2008; Carr and Friedman, al., 2006; Michalopoulos, 2007). However, in mammals, skeletal 1999; Hopkins, 1988), (Arnergard et al., 2010; Feulner muscle regeneration depends on a myogenic stem cell-dependent et al., 2009; Lovejoy et al., 2010), evolution of neural networks process without cell dedifferentiation (Zammit et al., 2006). These (Alves-Gomes, 2001; Bass, 1986; Zakon et al., 2008; Kawasaki, findings suggest that programs of tissue regeneration might be 2009; Arnegard et al., 2010), physiology of membrane excitability broadly conserved across these two vertebrate groups, but that their and synaptic plasticity (Bell et al., 2005; Gómez et al., 2005; Lewis expression is regulated by tissue-specific constraints. and Maler, 2004; Markham et al., 2009), and have been a source of material for the isolation of many molecules involved in these Vertebrate fish: regenerative capabilities biological processes. Not as well known is the considerable Adult bony fishes, unlike cartilaginous fishes, can completely regeneration capacity of South American gymnotiform electric regenerate fins and segments of liver and heart tissues (Table1). fishes. Weakly electric fish are among the most highly regenerative They can also regenerate almost any part of their central nervous species known (Table1; PubMed has an incomplete listing of 63 system including parts of the retina, optic , sections of the beginning in 1969 – it does not include many publications outside brainstem and many, if not all, spinal cord axons (Table1). of the USA). All gymnotiforms that have been tested can regenerate Unfortunately, the majority of fish species that possess a high their tails following amputation. Specifically, they can replace lost regenerative capacity are not yet amenable to genetic molecular dermis, spinal cord, electroreceptors, skeleton, blood vessels, fins, approaches. Consequently, studies on these fish species are limited skeletal muscle and the muscle-derived electric organ in a near- in the extent to which they can address molecular and cellular perfect replication of tissue pattern and without scar formation mechanisms of regeneration. (Table1). Some gymnotiforms can replace all tissues lost after

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Table 1. Regeneration in adult teleost fishes Fish species Type of injury Regeneration Reference Danio rerio (zebrafish) Mechanical lesion of body wall muscle Skeletal muscle fiber types Rowlerson et al., 1997 Amputated caudal fin Caudal fin Lee et al., 2005 Cardiotoxin injury of skeletal muscle Skeletal muscle Seger et al., 2011 Optic nerve crush Retinal neurons and glia Ramachandran et al., 2010; Thummel et al., 2006; Bernhardt et al., 1996 Photoreceptor degeneration Rod and cone photoreceptors Vihtelic and Hyde, 2000 Spinal cord transection Motor neuron axon regeneration; spinal van Raamsdonk et al., 1998; Ogai et al., cord glia regeneration; spinal cord 2012; Goldshmit et al., 2012; Becker et motor neurons al., 1997 Amputation of caudal fin Mechano--sensory organs in fin Dufourcq et al., 2006 Heart ventricle resection (20%) Ventricular myocardial fibers Poss et al., 2002; Yu et al., 2010 Surgical cuts into the dorsomedial Cerebellar glia and axons Liu et al., 2004 region of the corpus cerebelli of the cerebellum Salaria pavo (peacock Pectoral fin amputation Pectoral fin Misof and Wagner, 1992 blenny) Gambusia (Haplochilus) Caudal and pectoral fin amputation Caudal and pectoral fins Khalil and Aziz, 1989 schoelleri (mosquito fish) Carassius auratus Tail fin amputation Tail fin Santamaría et al., 1996 (goldfish) Denervation of extraocular muscles Axons innervating extraocular muscles Scherer, 1986; Scott, 1977 Optic nerve crush Optic nerve axons Schmidt and Shashoua, 1988 Tilapia mossambica Tail fin amputation Tail fin Kemp and Park, 1970 (tilapia) Oreochromis niloticus Surgical skeletal muscle injury Skeletal muscle Camargo et al., 2004 (Nile tilapia) Salmo salar () Inflammation--induced cardiac injury Cardiac muscle regeneration Ferguson et al., 2005 Polypterus senegalus Amputation of pectoral fin Pectoral fin regeneration Cuervo et al., 2012 and P. ornatipinnis () Cyprinus carpio () Tail fin amputation Epidermis of tail fin regeneration Böckelmann et al., 2010 Denervation of extraocular muscles Axons innervating extraocular muscles Mark et al., 1970 Oncorhynchus mykiss Amputation of lobed tail fin Tail fin regeneration Alonso et al., 2000 (rainbow trout) Pantadon bucholzi resection Regeneration of mechanoreceptive cells Jørgensen, 1991 (butterfly fish) Kryptopterus bicirrhus Lateral line resection Regeneration of mechanoreceptive cells Jørgensen, 1991 (glass )

Gymnotiform electric fishes

Sternopygus macrurus Tail amputation Skeletal muscle; electric organ; spinal Weber et al., 2012; Patterson and Zakon, (yellow-stripe knifefish) cord; skin; cartilage; blood vessels 1993. Sternopygus macrurus Removal of cheek skin Skin and electroreceptors Zakon, 1986; Fritzsch et al., 1990 (yellow--stripe knifefish) Toxin ablation of the supraorbital branch Axon regeneration Lanoo et al., 1993 leptorhynchus (brown of the anterior lateral line nerve ) Surgical lesions in cerebellum Cerebellar neurons and glia Zupanc, 2001 Spinal cord transection Spinal cord neurons and axons Sîrbulescu et al., 2009; Zupank and Ott, 1999 Sternarchus albifrons Tail amputation Electric organ; spinal cord neurons and Anderson and Waxman, 1981; () axons; Schwann cells Anderson and Waxman, 1983a; Anderson and Waxman, 1983b; Anderson et al., 1984; Anderson et al., 1985; Waxman and Anderson, 1980; Waxman and Anderson, 1985; Baillet- -Durbin, 1978 virescens Sectioning of lateral line nerve Sensory cell regeneration Bensouilah and Denizot, 1994 () Tail amputation Electric organ; anal fin; skeletal muscle; Srivastava, 1978; Kirschbaum and blood vessels; spinal cord axons; Meunier, 2005 cartilage; vertebrae carapo Tail amputation Electric organ; skeletal muscle; spinal Baillet--Derbin, 1969 () cord axons; skin Note: this summary table provides a sampling of published studies on regeneration of teleost fish including electric fish.

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Fig.2. Tail regeneration in an adult South American gymnotiform Sternopygus macrurus after amputation. (A)An adult control fish. (B)A tail 24h after amputation. Note the wound epithelium with no scar formation. (C)A 1-week blastema formed at the base of the amputation plane (dashed line) that appears as a visible swelling at the end of the tail. Cell differentiation proceeds from proximal to distal regions of the blastema. (D)A 2-week blastema formed at the base of the amputation plane (dashed line) that appears as a visible swelling at the end of the tail. (E)A 3-week regenerated tail showing the epidermal layer that is indistinguishable from that of control fish. Panels A and E are reproduced from Unguez and Zakon (Unguez and Zakon, 2002). Panels C and D are reproduced from Kim et al. (Kim et al., 2008). repeated tail amputations, suggesting an inexhaustible regeneration have exploited the inexhaustible regenerative capacity of adult S. capacity in the adult (Unguez and Zakon, 1998). macrurus to study the effects of neural input on the differentiation of muscle fibers and their phenotypic conversion into Skeletal muscle and electric organ regeneration in the long- electrocytes. Recently, we have also begun to address some tailed knifefish, Sternopygus macrurus aspects of the cellular and molecular mechanisms underlying the We have studied tail regeneration in the adult gymnotiform regeneration of complex tail structures such as skeletal muscle electric fish Sternopygus macrurus (Unguez and Zakon, 1998; and the electric organ in this teleost fish. Unguez and Zakon, 2002; Weber et al., 2012). We use this model Tail regeneration in gymnotiforms has been most studied in S. system because skeletal muscle cells of electric fish express a macrurus (Fig.2) (Patterson and Zakon, 1993; Patterson and Zakon, unique ability to undergo changes in their biochemical and 1996; Patterson and Zakon, 1997; Unguez and Zakon, 1998; Unguez morphological properties and give rise to electrocytes, the and Zakon, 2002; Weber et al., 2012). Like all gymnotiforms studied current-producing cells of the electric organ (Bennett, 1971; Fox to date, tail regeneration in S. macrurus begins by epidermal cells and Richardson, 1978; Fox and Richardson, 1979), and we have covering the wound followed by formation of a blastema (Fig.2). begun to determine how the developmental program for striated Specifically, when the tip of the tail is amputated, epidermal cells at muscle has been altered to produce an electrogenic, non- the wound margin rapidly proliferate and cover the wound within contractile cell that retains a partial muscle phenotype (Unguez 24h (Fig.2B). After 6–8days, a blastema appears as a small swelling and Zakon, 1998; Zakon and Unguez, 1999; Unguez and Zakon, at the end of the tail (Fig.2C). At this stage, the blastema is generally 2002; Kim et al., 2004; Kim et al., 2008; Cuellar et al., 2006). We less than 4mm in length. Over the following 6–8days, this swelling

Fig.3. Myogenic stem cells in adult skeletal muscle and the electric organ of S. macrurus. Pax7 labels myogenic satellite cells in adult skeletal muscle and the electric organ. (A)Pax7-positive cells (red) co-label with laminin (green) in tissue cryosections (20mm thick). Arrows point to localization of Pax7. Abbreviations: EC, electrocyte; mm, muscle fiber; epi, epidermis. (B)Electron micrograph cross- sections of adult intact tail show that satellite cells (SC) unlike other nuclei (N) are found between the plasma membrane (arrow) and basal lamina in both electrocytes and muscle fibers. Reproduced from Weber et al. (Weber et al., 2012).

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electrocytes proliferated and contributed to the regeneration blastema. Although it was demonstrated that myogenic stem cells were activated following tail amputation, a more rigorous identification of proliferative cell types and their degree of contribution to the blastema was warranted. Hence, we began to study the cellular mechanisms underlying the repetitive regeneration of myogenic tissues in S. macrurus using ultrastructural analysis, immunolabeling detection of the adult muscle stem cells called satellite cells, and in vivo microinjection studies of high molecular weight cell lineage tracers into single muscle fibers or electrocytes. First, our ultrastructural studies verified the anatomical presence of myogenic satellite cells in both muscle and the electric organ

Fig.4. Activation of Pax7-positive cells in S. macrurus near amputation site. Portions of a longitudinal cryosection (20mm thick) from 7-day blastema co-labeled with anti-Pax7 (red) and anti-laminin (green) antibodies. White line shows the site of tail amputation. Abbreviations: EC, electrocyte; Ep, epithelium; m, muscle fiber. elongates to an average 7mm in length (Fig.2D), and after 20–22days, the regenerated tail is covered by pigmented epithelium that is indistinguishable from the tail of uncut fish (Fig.2A).

Activation of myogenic stem cells in response to tail amputation in adult S. macrurus A study by Patterson and Zakon in 1993 tested the contribution of local reserve stem cells in the tail to the regeneration blastema (Patterson and Zakon, 1993). Using light microscopy observations and 5′-bromodeoxyuridine (BrdU) incorporation studies, the authors concluded that existing stem cells associated with muscle fibers and

Fig.6. Distribution of Pax7-positive cells in regions that give rise to muscle fibers and electrocytes. (A)Spatial distribution of Pax7-positive cells in regeneration blastema. Confocal images of longitudinal cryosection (20mm thick) from 14-day blastema immunolabeled with anti-Pax7 and BrdU antibodies. Arrowheads point to Pax7-positive cells adjacent to the epithelium (Ep). Reproduced from Weber et al. (Weber et al., 2012). (B)Pax7-positive cells are localized in blastema regions that give rise to muscle, electric organ and dorsal spinal cord. Serial cross-sections taken from distal half of 14-day blastemas immunolabeled with antibodies against Pax7 (green) and myosin heavy chain (MyHC). MyHC is present in all Fig.5. Activation of Pax7-positive cells near intact electrocytes (EC). muscle fibers, which are small cells located between epithelium and Portions of longitudinal cryosection (20mm thick) from 14-day blastema co- developing electrocytes (ECs), which are larger cells more medially located labeled with anti-Pax7 (green) and anti-BrdU (red) antibodies. White (arrows). Co-labeling by anti-Pax7 and anti-MyHC antibodies was detected dashed line shows the site of tail amputation. Arrows point to cells that in peripheral regions of the blastema underneath the epithelium. Pax7 were co-labeled with Pax7 and BrdU. Reproduced from Weber et al. labeling was also detected in dorsal spinal cord (SC). Reproduced from (Weber et al., 2012). Weber et al. (Weber et al., 2012).

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(Fig.3). Second, because Pax7 is a commonly used marker for muscle fibers were labeled with single-cell dextran injections prior identifying quiescent satellite cells in mammals (Seale et al., 2000; to tail amputation. Tails analyzed up to 14days post-amputation Zammit et al., 2004), we carried out molecular and immunolabeling showed an intact morphology of these myogenic cells with no studies of Pax7 expression in S. macrurus tissues. These studies fragmentation into smaller cellular components (Fig.7), suggesting showed that Pax7-positive cells were associated with electrocytes that skeletal muscle or electric organ dedifferentiation did not occur. and muscle fibers of intact adult tails (Fig.3). By combining nuclear In a separate group of fish, serial longitudinal cryosections were Pax7 expression with ultrastructural analysis of satellite cells, we immunolabeled with laminin and morphological analysis did not concluded that Pax7 is a reliable satellite cell marker for S. reveal any evidence of electrocyte or muscle fiber fragmentation macrurus. By virtue of their nuclear Pax7 expression, morphology (Weber et al., 2012). Although these data do not negate the possibility and location, these cells associated with muscle fibers and that some cell dedifferentiation does take place, they do not support electrocytes were considered to be homologs of satellite cells cell dedifferentiation as the essential mechanism underlying the observed in mammals (Schultz et al., 2006; Ishido et al., 2009), robust regeneration capacity of myogenic tissues in S. macrurus. salamanders (Morrison et al., 2006), frogs (Chen et al., 2006) and Instead, together with our Pax7 analyses, our studies provide support other (Steinbacher et al., 2007). Moreover, these data for the occurrence of a stem cell activation mechanism for provided the first example of Pax7-positive muscle progenitor cells epimorphic regeneration in this vertebrate species. localized within a non-contractile electrogenic tissue, representing an exception to the tissue specificity of these myogenic stem cells Conclusions and future directions to striated muscle fibers in vertebrates. Regeneration is not merely a curious coincidence, it highlights a We then studied the response of Pax7-positive cells following biologically relevant feature that has been selected for in both amputation, blastema formation, and regeneration of muscle fibers terrestrial and aquatic environments. Gymnotiform fishes, like and electrocytes over a 2week period. Upon tail amputation, we urodele amphibians, possess a large capacity as adults to fully detected an increase in the number of Pax7-positive cells that regenerate injured or amputated body parts that contain multiple associated with intact electrocytes and muscle fibers near the tissue types, including excised portions of spinal cord and skeletal amputation site (Fig.4). This increase in the number of Pax7- muscle. Data from our Pax7 expression studies in combination with positive cells continued up to 14days after tail amputation, at which intracellular dye injection experiments are significant because they time their density was markedly higher (Fig.5) than that found in reveal that in the adult S. macrurus, restoration of skeletal muscle control tissues (Fig.3). Within the regeneration blastema, Pax7- and the muscle-derived electric organ relies on the activation of positive cells were found exclusively in regions where muscle myogenic stem cells for the renewal of both myogenic tissues. fibers and electrocytes form de novo (Fig.6A), as demonstrated by These data are consistent with the emergent concept in vertebrate the co-labeling of myogenic markers for sarcomeric myosin heavy regeneration that different tissues are sources of distinct progenitor chain expression (Fig.6B). Further, these myogenic precursor cells, cell populations to the regeneration blastema, and these progenitor at least based on their compartmentalization in the regeneration cells subsequently restore the original tissue (Tanaka and Reddien, blastema, did not seem to contribute to tissues other than skeletal 2011). In conclusion, cell dedifferentiation is likely one muscle and the electric organ. For these reasons, our results suggest regeneration strategy in adult S. macrurus, but it is not a that muscle and electrocyte regeneration in adult S. macrurus is requirement in the regeneration of skeletal muscle and the muscle- similar to the satellite-dependent muscle repair process common in derived electric organ. Similarly, the incidence of tissue-specific vertebrates, including mammals, frogs, birds, fish and some stem cells contributing to the blastema in adults has been reported salamanders (Hawke and Garry, 2001; Zammit et al., 2004; in limb regeneration in urodele amphibians (Morrison et al., 2006) Morrison et al., 2006; Morrison et al., 2010; Feldman et al., 1993). and antler regeneration in deer (Li et al., 2007). These reports emphasize the significance of investigating a broad range of species No evidence for myogenic cell dedifferentiation after tail in order to expand our knowledge on the different strategies used amputation in adult S. macrurus to restore lost tissues and assess the extent to which the identified As in previous urodele regeneration experiments, we also performed cellular and molecular mechanisms that produce new cells are in vivo microinjection studies using high molecular weight lineage conserved across different groups. One could imagine that tracers to test whether the source of proliferating cells proximal to inclusion of a wider range of species in regeneration studies may the amputation site were mature cells that underwent generate novel information useful to modify our current model of dedifferentiation. In these experiments, intact electrocytes and epimorphic regeneration in vertebrates.

Fig.7. Intracellular tracer dye injections into single muscle fibers and electrocytes at the level of the distal-most ventral fin reveal no cell fragmentation. Fluororuby (red) was injected into single electrocytes (A) and muscle fibers (B) and visualized 7days after injection by retracting the overlying skin of the fish. Tail sections at the level of the distal-most region of the ventral fin were processed for immunolabeling and viewed under a fluorescent microscope. (C)Region of a cryosection containing muscle fibers injected with Fluororuby Dextran (red) and counterstained with DAPI (blue). (D)Same image as C showing only DAPI labeling. Scale bars: (A) 20mm; (D) 10mm. Reproduced from Weber et al. (Weber et al., 2012).

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Fig.8. Tail regeneration in adult gymnotiforms and Apteronotus albifrons following amputation. Left panels: adult control Eigenmannia virescens (glass knifefish) and Apteronotus albifrons (black ghost knifefish). In both gymnotiforms, regeneration proceeds through the formation of a blastema at the base of the amputation plane (dashed line) and cell differentiation occurs from proximal to distal regions of the blastema. Right panels: regenerated tails in Eigenmannia virescens and Apteronotus albifrons 2weeks and 12months after tail cut, respectively. The 2-week regeneration blastema in E. virescens (top right panel) parallels that observed in S. macrurus and its subsequent regeneration results in the restoration of all tissues and structures in proportions comparable to that of an intact adult tail. In A. albifrons, regeneration after amputation proceeds with blastema formation (not shown) and differentiation of anal-like fin structure (arrow) with stunted tail segment. Photo credit: Vincent Gutschick.

Our studies using S. macrurus also underline the significance of in muscle regeneration. Addressing these issues in a model system determining whether the Pax7-positive cells in muscle and the such as S. macrurus, where satellite cells associate with muscle electric organ form a single multipotent class of cells or whether fibers and electrocytes, which differ significantly in nerve- they represent discrete satellite cells with different tissue-specific dependent activation patterns (Mills et al., 1992), would provide fates and replication capacities following injury. For example, it important insight into the roles that defined environmental cues will be interesting to determine whether Pax7-positive cells in adult play in the satellite cell biological phenotype. myogenic tissues reflect their cell type of origin during the The South American gymnotiform fishes are monophyletic but recapitulation of tissue organization after tail amputation, as has comprise a considerably diversified group (Albert and Crampton, been observed with satellite cell populations that give rise to either 2006). Although closely related, different gymnotiform species slow or fast muscle fibers in birds and rodents (Feldman and exhibit diversity in their general morphology and anatomy, size and Stockdale, 1991; Rosenblatt et al., 1996). Differences in replication shape of their individual electrocytes, and shape and duration of capacities between satellite cells associated with muscle fibers their electric organ discharge signals (Bennett, 1971; Albert and versus cells of the electric organ, if present, would also be Crampton, 2006). It is important to note that adults also vary consistent with findings from mammalian satellite cell studies considerably in the extent to which they restore the complexity and (Collins et al., 2007; Zammit et al., 2006; Zammit, 2008; Kuang et structural organization of lost tissues after tail amputation. For al., 2007). One way to explore this is to replicate differentiation of example, the glass knifefish Eigenmannia virescens regenerates its muscle fibers and electrocytes in culture. Our ability to isolate tail following amputation much like S. macrurus, i.e. a regeneration satellite cells from muscle fibers or electrocytes will allow us to blastema forms, grows and restores the different tissues of the tail directly test the potential of these myogenic cells under suitable in a structural organization resembling that of an uncut tail (Figs2, differentiation conditions (Shadd et al., 2008). Future studies can 8). Interestingly, a comparable tail amputation in the adult black help identify specific environmental cues that may influence the ghost knifefish, Apteronotus albifrons, proceeds with formation of proliferative and differentiation potentials of satellite cells a regeneration blastema, but its growth and differentiation results associated with muscle and the electric organ. in a stunted tail with an anal fin-like structure at the distal end The effectiveness of satellite cells in restoring damaged muscle (Fig.8). To what extent are similar mechanisms to regenerate in vertebrates is regulated by the interplay between differences in myogenic tissue at work? And what factors determine the type and the inherent potential of the satellite cells themselves and the shape of tissues that comprise the tail regenerate in A. albifrons? external cues from the surrounding environment (Dhawan and Experimental approaches (intracellular dye microinjections) and Rando, 2005; Collins et al., 2007). In this regard, studies have availability of useful markers (cell type markers) that can be applied reported that muscle regeneration in adult mammals can initially across close relatives will be essential in comparing molecular proceed to some extent in the absence of innervation, but growth factors and cellular processes underlying regeneration among and terminal differentiation of regenerated fibers and continued gymnotiforms. In sum, our findings in S. macrurus regeneration regeneration is impeded in denervated muscles beyond a week after tail amputation not only raise important questions regarding (Mussini et al., 1987; Sesodia and Cullen, 1991). Whether this the extent to which a myogenic stem cell-dependent regeneration ineffective muscle regeneration is due to changes in neural factors process is conserved and active in vertebrate species other than that inhibit the regenerative ability of otherwise competent satellite urodele amphibians and zebrafish, but also warrant further cells is not known. Relatively little work has been carried out to investigations to determine why and how regenerative abilities date on the potential influences of neuronal factors (electrical differ among the closely related species of gymnotiform electric activity or activity-independent) on satellite cells and early events fish.

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