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

Biology is replete with examples of regeneration, the process that allows animals to replace or repair cells, tissues and organs. Regeneration provides obvious benefits to survival and function and is known to occur widely among animal phyla (Bely, 2010; Sánchez Alvarado and Tsonis, 2006). Some animals perform this remarkable feat almost flawlessly. However, the ability to fully regenerate complex tissues decreases as one moves from the basal groups in the animal kingdom to those more highly derived groups (Goss, 1969). For example, invertebrates including jellyfish, planarians, segmented worms, mollusks and starfish can fully regenerate multiple tissues and even entire body parts (Brusca and Brusca, 1990; Ferretti and Geraudie, 1998; Brockes and Kumar, 2002). Among vertebrates, amphibians and fishes generally express a robust regeneration response to limb loss, whereas reptiles, birds and mammals have a more limited capacity to regenerate complex body structures (Brockes and Kumar, 2008; Galis et al., 2003). The evolutionary loss of regenerative ability in some groups, to varying degrees, remains an unsolved problem that attracts many biologists (Bely and Nyberg, 2010). And, not surprisingly, the limited capacity of humans to regenerate and restore tissues and organs fuels enormous interest from a medical perspective in elucidating the mechanisms responsible for the extensive regenerative capacities in vertebrates.

In general, studies exploring regeneration show that vertebrate species that can restore amputated limb structures during early development exhibit little to no regenerative capacity as adults. For example, all adult frogs either do not regenerate their limbs at all or they regenerate a spike of cartilage surrounded by skin – an ‘outgrowth’, but hardly a limb – whereas all immature frogs or ‘tadpoles’ easily regenerate their limbs (Muneoka et al., 1986). Progressive decline in the regenerative response to limb amputation with increasing age has also been demonstrated in rodents (Deuchar, 1976; Lee and Chan, 1991; Wanek et al., 1989). In mammals, skeletal muscle is considered a highly regenerative tissue as it can repair itself after injury or disease. Similar to limb regeneration, aging skeletal muscle shows a diminished ability to repair itself and an increased susceptibility to contraction-induced injury (Alnaqeeb and Goldspink, 1987; Brooks and Faulkner, 1994). Failure to repair a damaged region of muscle fiber can result in severe cell atrophy and even cell death. This is in stark contrast to the inexhaustible regeneration of skeletal muscle tissues and even entire body parts exhibited by some adult non-mammalian species (Unguez and Zakon, 1998; Kirschbaum and Meunier, 1988; Sánchez Alvarado and Tsonis, 2006).

Regeneration processes are grouped according to the presence or absence of cell division during the replacement of lost tissues (Morgan, 1901). Morphallaxis occurs in the absence of cell proliferation, and epimorphosis requires cell proliferation. Epimorphosis is further categorized according to whether a regeneration blastema forms. A blastema is a specialized proliferative zone of mesenchymal cells that forms beneath the epidermis at the wound site. Blastema formation is a key process in epimorphic regeneration in both invertebrates and vertebrates (Thouveny and Tassava, 1998). The widespread distribution of this feature across animal taxa supports the hypothesis that a program for epimorphic regeneration may have been conserved through evolution (Brusca and Brusca, 1990; Sanchez-Alvarado, 2000). To date, the general notion is that a blastema is formed primarily from the re-entry of mature cells near the injury site into the cell cycle, i.e. dedifferentiation, to contribute to the restoration of lost tissue. In fact, the idea that a greater potential for epimorphic regeneration is associated with a corresponding potential for cell dedifferentiation remains broadly accepted (Brockes and Kumar, 2002; Nye et al., 2003; Tsonis, 1991). Support for this idea is based largely on the vast body of evidence from experiments in urodele amphibians such as the newt and axolotls, which are able to regenerate limbs, tail, jaws, ocular tissues including retina and lens, and some portions of the heart. Specifically, in vivo cell tracing experiments in urodele amphibians have shown that mature cells including muscle fibers can dedifferentiate, proliferate and enter the blastema (Stocum, 1995; Echeverri et al., 2001; Brockes and Kumar, 2002; Nye et al., 2003; Nechiporuk and Keating, 2002; Tanaka and Reddien, 2011). Similarly, the robust regenerative capacity of zebrafish to regrow amputated fins, heart and neural tissue such as the spinal cord and retina is suggested to depend on dedifferentiation of cells near the amputation site by studies using transgenesis, loss- and gain-of-function technology, and cell labeling methodology (Tanaka and Reddien, 2011; Singh et al., 2012; Poss et al., 2003; Poss, 2010).

Although cell dedifferentiation might be favored under certain conditions, it is not possible to exclude the contribution of alternative modes of regeneration. For example, generation of reliable cell markers have led to recent findings that resident, tissue-specific stem cells also respond to injury and contribute to blastema formation in some vertebrate species (Li et al., 2007; Morrison et al., 2006; Poss et al., 2003; Singh et al., 2012; Weber et al., 2012). Hence, the extent to which cell dedifferentiation versus stem cell activation occurs in response to injury in vertebrates warrants further investigation. Interestingly, multiple regeneration responses may come into play following injury or disease in the same animal. In mammals, as in zebrafish, liver regeneration is accomplished largely by hepatocyte dedifferentiation (Kan et al., 2009; Fausto et al., 2006; Michalopoulos, 2007). However, in mammals, skeletal muscle regeneration depends on a myogenic stem cell-dependent process without cell dedifferentiation (Zammit et al., 2006). These findings suggest that programs of tissue regeneration might be broadly conserved across these two vertebrate groups, but that their expression is regulated by tissue-specific constraints.

Adult bony fishes, unlike cartilaginous fishes, can completely regenerate fins and segments of liver and heart tissues (Table 1). They can also regenerate almost any part of their central nervous system including parts of the retina, optic nerve, sections of the brainstem and many, if not all, spinal cord axons (Table 1). Unfortunately, the majority of fish species that possess a high regenerative capacity are not yet amenable to genetic molecular approaches. Consequently, studies on these fish species are limited in the extent to which they can address molecular and cellular mechanisms of regeneration.

Research in fish regeneration biology has focused largely on the zebrafish, as it possesses a large regenerative capacity and is an ideal model system for investigation using cellular, molecular and genetic approaches and (Poss et al., 2003; Akimenko et al., 2003). Studies on zebrafish regeneration are contributing extensively to our current understanding of regeneration mechanisms to replace different cell and tissue types. Indeed, the ability to carry out loss- and gain-of-function experiments in combination with genetic screens and in vivo cell tracing is increasing the identification of regulatory factors and progenitor cells involved in zebrafish regeneration (Tanaka and Reddien, 2011; Sánchez Alvarado and Tsonis, 2006; Singh et al., 2012). Even so, the ability of adult zebrafish to fully repair and restore all damaged or lost tissues, organs and limbs is limited. For example, a zebrafish cannot regenerate its tail if amputation occurs proximal to the caudal fin (Fig. 1). In contrast, many South American gymnotiform electric fish species can repeatedly regenerate their tails following amputation during adulthood.

There are more than 700 vertebrate species known that are capable of generating electricity and all of them are fishes representing 11 independent lineages (Albert and Crampton, 2006). Of these, approximately 173 species are gymnotiforms, or South American knifefishes, and in all but one group, the electric organ derives from striated muscle cells that suppress many muscle phenotypic properties (Bennett, 1971). Gymnotiforms have been used to study the formation of sensory and motor specializations (Fox and Richardson, 1978; Fox and Richardson, 1979; Kirschbaum and Schwassmann, 2008; Denizot et al., 1998; Denizot et al., 2007; Vischer, 1989a; Vischer, 1989b; Zakon, 1984; Lannoo et al., 1990), animal communication (Zakon et al., 2008; Carr and Friedman, 1999; Hopkins, 1988), speciation (Arnergard et al., 2010; Feulner et al., 2009; Lovejoy et al., 2010), evolution of neural networks (Alves-Gomes, 2001; Bass, 1986; Zakon et al., 2008; Kawasaki, 2009; Arnegard et al., 2010), physiology of membrane excitability and synaptic plasticity (Bell et al., 2005; Gómez et al., 2005; Lewis and Maler, 2004; Markham et al., 2009), and have been a source of material for the isolation of many molecules involved in these biological processes. Not as well known is the considerable regeneration capacity of South American gymnotiform electric fishes. Weakly electric fish are among the most highly regenerative species known (Table 1; PubMed has an incomplete listing of 63 beginning in 1969 – it does not include many publications outside of the USA). All gymnotiforms that have been tested can regenerate their tails following amputation. Specifically, they can replace lost dermis, spinal cord, electroreceptors, skeleton, blood vessels, fins, skeletal muscle and the muscle-derived electric organ in a near-perfect replication of tissue pattern and without scar formation (Table 1). Some gymnotiforms can replace all tissues lost after repeated tail amputations, suggesting an inexhaustible regeneration capacity in the adult (Unguez and Zakon, 1998).

We have studied tail regeneration in the adult gymnotiform electric fish Sternopygus macrurus (Unguez and Zakon, 1998; Unguez and Zakon, 2002; Weber et al., 2012). We use this model system because skeletal muscle cells of electric fish express a unique ability to undergo changes in their biochemical and morphological properties and give rise to electrocytes, the current-producing cells of the electric organ (Bennett, 1971; Fox and Richardson, 1978; Fox and Richardson, 1979), and we have begun to determine how the developmental program for striated muscle has been altered to produce an electrogenic, non-contractile cell that retains a partial muscle phenotype (Unguez and Zakon, 1998; Zakon and Unguez, 1999; Unguez and Zakon, 2002; Kim et al., 2004; Kim et al., 2008; Cuellar et al., 2006). We have exploited the inexhaustible regenerative capacity of adult S. macrurus to study the effects of neural input on the differentiation of muscle fibers and their phenotypic conversion into electrocytes. Recently, we have also begun to address some aspects of the cellular and molecular mechanisms underlying the regeneration of complex tail structures such as skeletal muscle and the electric organ in this teleost fish.

Tail regeneration in gymnotiforms has been most studied in S. macrurus (Fig. 2) (Patterson and Zakon, 1993; Patterson and Zakon, 1996; Patterson and Zakon, 1997; Unguez and Zakon, 1998; Unguez and Zakon, 2002; Weber et al., 2012). Like all gymnotiforms studied to date, tail regeneration in S. macrurus begins by epidermal cells covering the wound followed by formation of a blastema (Fig. 2). Specifically, when the tip of the tail is amputated, epidermal cells at the wound margin rapidly proliferate and cover the wound within 24 h (Fig. 2B). After 6–8 days, a blastema appears as a small swelling at the end of the tail (Fig. 2C). At this stage, the blastema is generally less than 4 mm in length. Over the following 6–8 days, this swelling 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).

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 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. 3). Second, because Pax7 is a commonly used marker for identifying quiescent satellite cells in mammals (Seale et al., 2000; Zammit et al., 2004), we carried out molecular and immunolabeling studies of Pax7 expression in S. macrurus tissues. These studies showed that Pax7-positive cells were associated with electrocytes and muscle fibers of intact adult tails (Fig. 3). By combining nuclear Pax7 expression with ultrastructural analysis of satellite cells, we concluded that Pax7 is a reliable satellite cell marker for S. macrurus. By virtue of their nuclear Pax7 expression, morphology and location, these cells associated with muscle fibers and electrocytes were considered to be homologs of satellite cells observed in mammals (Schultz et al., 2006; Ishido et al., 2009), salamanders (Morrison et al., 2006), frogs (Chen et al., 2006) and other teleosts (Steinbacher et al., 2007). Moreover, these data provided the first example of Pax7-positive muscle progenitor cells localized within a non-contractile electrogenic tissue, representing an exception to the tissue specificity of these myogenic stem cells to striated muscle fibers in vertebrates.

We then studied the response of Pax7-positive cells following amputation, blastema formation, and regeneration of muscle fibers and electrocytes over a 2 week period. Upon tail amputation, we detected an increase in the number of Pax7-positive cells that associated with intact electrocytes and muscle fibers near the amputation site (Fig. 4). This increase in the number of Pax7-positive cells continued up to 14 days after tail amputation, at which time their density was markedly higher (Fig. 5) than that found in control tissues (Fig. 3). Within the regeneration blastema, Pax7-positive cells were found exclusively in regions where muscle fibers and electrocytes form de novo (Fig. 6A), as demonstrated by the co-labeling of myogenic markers for sarcomeric myosin heavy chain expression (Fig. 6B). Further, these myogenic precursor cells, at least based on their compartmentalization in the regeneration blastema, did not seem to contribute to tissues other than skeletal muscle and the electric organ. For these reasons, our results suggest that muscle and electrocyte regeneration in adult S. macrurus is similar to the satellite-dependent muscle repair process common in vertebrates, including mammals, frogs, birds, fish and some salamanders (Hawke and Garry, 2001; Zammit et al., 2004; Morrison et al., 2006; Morrison et al., 2010; Feldman et al., 1993).

As in previous urodele regeneration experiments, we also performed in vivo microinjection studies using high molecular weight lineage tracers to test whether the source of proliferating cells proximal to the amputation site were mature cells that underwent dedifferentiation. In these experiments, intact electrocytes and muscle fibers were labeled with single-cell dextran injections prior to tail amputation. Tails analyzed up to 14 days post-amputation showed an intact morphology of these myogenic cells with no fragmentation into smaller cellular components (Fig. 7), suggesting that skeletal muscle or electric organ dedifferentiation did not occur. In a separate group of fish, serial longitudinal cryosections were immunolabeled with laminin and morphological analysis did not reveal any evidence of electrocyte or muscle fiber fragmentation (Weber et al., 2012). Although these data do not negate the possibility that some cell dedifferentiation does take place, they do not support cell dedifferentiation as the essential mechanism underlying the robust regeneration capacity of myogenic tissues in S. macrurus. Instead, together with our Pax7 analyses, our studies provide support for the occurrence of a stem cell activation mechanism for epimorphic regeneration in this vertebrate species.

Regeneration is not merely a curious coincidence, it highlights a biologically relevant feature that has been selected for in both terrestrial and aquatic environments. Gymnotiform fishes, like urodele amphibians, possess a large capacity as adults to fully regenerate injured or amputated body parts that contain multiple tissue types, including excised portions of spinal cord and skeletal muscle. Data from our Pax7 expression studies in combination with intracellular dye injection experiments are significant because they reveal that in the adult S. macrurus, restoration of skeletal muscle and the muscle-derived electric organ relies on the activation of myogenic stem cells for the renewal of both myogenic tissues. These data are consistent with the emergent concept in vertebrate regeneration that different tissues are sources of distinct progenitor cell populations to the regeneration blastema, and these progenitor cells subsequently restore the original tissue (Tanaka and Reddien, 2011). In conclusion, cell dedifferentiation is likely one regeneration strategy in adult S. macrurus, but it is not a requirement in the regeneration of skeletal muscle and the muscle-derived electric organ. Similarly, the incidence of tissue-specific stem cells contributing to the blastema in adults has been reported in limb regeneration in urodele amphibians (Morrison et al., 2006) and antler regeneration in deer (Li et al., 2007). These reports emphasize the significance of investigating a broad range of species in order to expand our knowledge on the different strategies used to restore lost tissues and assess the extent to which the identified cellular and molecular mechanisms that produce new cells are conserved across different groups. One could imagine that inclusion of a wider range of species in regeneration studies may generate novel information useful to modify our current model of epimorphic regeneration in vertebrates.

Our studies using S. macrurus also underline the significance of determining whether the Pax7-positive cells in muscle and the electric organ form a single multipotent class of cells or whether they represent discrete satellite cells with different tissue-specific fates and replication capacities following injury. For example, it will be interesting to determine whether Pax7-positive cells in adult myogenic tissues reflect their cell type of origin during the recapitulation of tissue organization after tail amputation, as has been observed with satellite cell populations that give rise to either slow or fast muscle fibers in birds and rodents (Feldman and Stockdale, 1991; Rosenblatt et al., 1996). Differences in replication capacities between satellite cells associated with muscle fibers versus cells of the electric organ, if present, would also be consistent with findings from mammalian satellite cell studies (Collins et al., 2007; Zammit et al., 2006; Zammit, 2008; Kuang et al., 2007). One way to explore this is to replicate differentiation of muscle fibers and electrocytes in culture. Our ability to isolate satellite cells from muscle fibers or electrocytes will allow us to directly test the potential of these myogenic cells under suitable differentiation conditions (Shadd et al., 2008). Future studies can help identify specific environmental cues that may influence the proliferative and differentiation potentials of satellite cells associated with muscle and the electric organ.

The effectiveness of satellite cells in restoring damaged muscle in vertebrates is regulated by the interplay between differences in the inherent potential of the satellite cells themselves and the external cues from the surrounding environment (Dhawan and Rando, 2005; Collins et al., 2007). In this regard, studies have reported that muscle regeneration in adult mammals can initially proceed to some extent in the absence of innervation, but growth and terminal differentiation of regenerated fibers and continued regeneration is impeded in denervated muscles beyond a week (Mussini et al., 1987; Sesodia and Cullen, 1991). Whether this ineffective muscle regeneration is due to changes in neural factors that inhibit the regenerative ability of otherwise competent satellite cells is not known. Relatively little work has been carried out to date on the potential influences of neuronal factors (electrical activity or activity-independent) on satellite cells and early events in muscle regeneration. Addressing these issues in a model system such as S. macrurus, where satellite cells associate with muscle fibers and electrocytes, which differ significantly in nerve-dependent activation patterns (Mills et al., 1992), would provide important insight into the roles that defined environmental cues play in the satellite cell biological phenotype.

The South American gymnotiform fishes are monophyletic but comprise a considerably diversified group (Albert and Crampton, 2006). Although closely related, different gymnotiform species exhibit diversity in their general morphology and anatomy, size and shape of their individual electrocytes, and shape and duration of their electric organ discharge signals (Bennett, 1971; Albert and Crampton, 2006). It is important to note that adults also vary considerably in the extent to which they restore the complexity and structural organization of lost tissues after tail amputation. For example, the glass knifefish Eigenmannia virescens regenerates its tail following amputation much like S. macrurus, i.e. a regeneration blastema forms, grows and restores the different tissues of the tail in a structural organization resembling that of an uncut tail (Figs 2, 8). Interestingly, a comparable tail amputation in the adult black ghost knifefish, Apteronotus albifrons, proceeds with formation of a regeneration blastema, but its growth and differentiation results in a stunted tail with an anal fin-like structure at the distal end (Fig. 8). To what extent are similar mechanisms to regenerate myogenic tissue at work? And what factors determine the type and shape of tissues that comprise the tail regenerate in A. albifrons? Experimental approaches (intracellular dye microinjections) and availability of useful markers (cell type markers) that can be applied across close relatives will be essential in comparing molecular factors and cellular processes underlying regeneration among gymnotiforms. In sum, our findings in S. macrurus regeneration after tail amputation not only raise important questions regarding the extent to which a myogenic stem cell-dependent regeneration process is conserved and active in vertebrate species other than urodele amphibians and zebrafish, but also warrant further investigations to determine why and how regenerative abilities differ among the closely related species of gymnotiform electric fish.

Live fish photography by Vince Gutschick and Michael McDowell. Dr Laura Burrus provided insightful comments on the manuscript.