Transdifferentiation of Muscle to Electric Organ: Regulation of Muscle-Specific Proteins Is Independent of Patterned Nerve Activity

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DEVELOPMENTAL BIOLOGY 186, 115-126 (1997) ARTICLE NO. DB978580 Transdifferentiation of Muscle to Electric Organ: Regulation of Muscle-Specific Proteins Is Independent of Patterned Nerve Activity

John M. Patterson I and Harold H. Zakon 2 Department of Zoology and Center for Developmental Biology, University of Texas at Austin, Austin, Texas 78712

Transdifferentiation is the conversion of one differentiated cell type into another. The electric organ of fishes transdifferen- tiates from muscle but little is known about how this occurs. To begin to address this question, we studied the expression of muscle- and electrocyte-specific proteins with immunohistochemistry during regeneration of the electric organ. In the early stages of regeneration, a blastema forms. Blastemal ceils cluster, express desmin, fuse into myotubes, and then express tr-actinin r tropomyosin, and myosin. Myotubes in the periphery of the blastema continue to differentiate as muscle; those in the center grow in size, probably by fusing with each other, and lose their sarcomeres as they become electrocytes. Tropomyosin is rapidly down.regulated while desmin, tr-actinin, and myosin continue to be diffusely expressed in newly formed electrocytes despite the absence of organized sarcomeres. During this time an isoform of keratin that is a marker for mature electrocytes is expressed. One week later, the immunoreactivities of myosin disappears and t~-actinin weakens, while that of desmin and keratin remain strong. Since nerve fibers grow into the blastema preceding the appearance of any differentiated cells, we tested whether the highly rhythmic nerve activity associated with electromotor input plays a role in transdifferentiation and found that electrocytes develop normally in the absence of electromotor neuron activity. © 1997 Academic Press

INTRODUCTION 1972; Szabo, 1960). In some species, such as the mormyrid teleost Gymnarchus and the elasmobranch Raja, differenti- During evolution of the fishes, the electric organ (EO) has ated electrocytes still contain sarcomeres, although they arisen independently at least seven times, in each case from may be disrupted (Bennett, 1971; Schwartz et al., 1975). In muscle (Bass, 1986; Zakon, 1995). Yet, we know little about others, such as the gymnotiforms Sternopygus and Eigen- what predisposes muscle to form EO. In all species exam- mannia or the elasmobranch Torpedo, no sign of organized ined to date, electrocytcs arise from a pool of myoblasts myofibrils is present in mature cells (Bennett, 19711. otherwise identical to those that give rise to muscle during In the weakly electric gymnotiforms, one of the most the earliest stages of myogenesis (Dahlgren, 1911; Keynes, highly regenerative groups of vertebrates, electrocytes re- 1961; Wachtel, 1964; Fox and Richardson, 1978; Neville and generate after partial amputation of the EO. Regenerating Schmidt, 1992; Patterson and Zakon, 1993). They are in electrocytes in this group show the same pattern of forma- contact with ingrowing electromotor nerves (Srivatastava tion through fusion of myoblasts and transient expression and Szabo, 1972; Fox and Richardson, 1978; La Rochelle et of sarcomeres followed by myofibrdlar breakdown as do al., 1990), although the role of the innervation in establish- developing electrocytes (Eigenrnannia: Baillet-Derbin, 1978; ing phenotype is not known. Sternopygus: Patterson, unpublished results). As they differentiate further, electrocytes develop myo- Previous work using electron microscopy (Baillet-Derbin, fibrils (Fox and Richardson, 1978; Srivatastava and Szabo, 1978) provided clues to the profound changes in the ultra- structural makeup of these cells but did not allow for identi- Present address: UIC Department of Biological Sciences, SES fication of specific muscle or electrocyte proteins. Based on Bldg., 845 W. Taylor St., Chicago, IL 60607-7060. the immunocytochemical profile of proteins in the mature To whom correspondence and reprint requests should be ad- electrocytes and muscles in the electric organ of the gym- dressed. Fax: (512) 471-9651. E-mail: [email protected]. notiforms Sternopygus macrurus (Patterson and Zakon,

1996), we examined the conversion of electrocytes from non blastema. Fish were reanesthetized and the blastema was ex- muscle during electric organ regeneration in this species as amined at Days 7, 10, and 14. a first step in a molecular analysis of this process. First, we determined whether a number of representative sarcomeric proteins are coordinately or independently regulated and when electrocyte-specific markers appear relative to the RESULTS changes in sarcomeric proteins. Second, since the motorneurons that innervate the electric organ constantly Overview of Regeneration in the Electric Organ drive it at rates of 50-200 Hz in this species (Zakon, 1996), we tested whether this regular patterned electrical activity The process of regeneration moves in a proximodistal di- was necessary for changes in expression of sarcomeric pro- rection in the blastema (Fig. 1). Therefore, at later times, it teins and the appearance of the electrocyte phenotype. was possible to see all stages of regrowth within one tail. For example, by Day 12 or 14 while newly formed electrocytes were in the final stages of maturation close to the wound margin, in the more distal parts of the blastema, MATERIALS AND METHODS early myogenic events were snll occurring. We will focus on the series of events that occur in blastemas of the 12- and Fish. S. rnacrurus were obtained from a commercial fish im- 14-day fish and supplement that with data from blastemas porter (Z-Fish, Los Angeles, CA). Fish of both sexes, 20-35 era, taken earlier in the regeneration process when necessary to were used. Tank temperature was maintained at 26-28°C. establish the time of occurrence of earlier events. lmmunohistochemistry. Eighteen fish were anesthetized in EO amputation induces a series of repair mechanisms MS-222 {1:10,000 in tank water) and a 1-cm portion of EO was that proceed in two steps: damaged tissue dies and is re- removed. After 4, 6, 8, 10, 12, and 14 days of regrowth (N = 3 fish moved while progeny of satellite cells from EO and muscle per time point), the fish were reanesthetized and a portion of the EO containing the stump of old tissue plus any regeneration blas- invade the site of damage, proliferate to form a blastema, tema was removed from three fish at each time point. EO tissue and differentiate into new structures. We found that electro- was immediately frozen at -80°C in isopentane and 1S-ram sec- cytes transected during amputation collapsed and finally tions were cut (longitudinal and cross sections) and mounted on degenerated by Day 8 (Fig. 1). By Day 4 large numbers of subbed slides. Sections were blocked with 0.2% bovine serum albu- undifferentiated mesenchymal cells were multiplying at the min (BSA) in 0.1 M PBS for 30 rain. Sections were then incubated wound site and began forming a visible blastema on Day 6. overnight at 4°C with one of the following monoclonal antibodies The first immunocytochemical indication of myogenesis (see Patterson and Zakon, 1996, for details): MF20 (anti-myosin also occurred on Days 6-8. By Day 10 the first immunocyto- heavy chain); D76 (anti-desmin); 88b (anti-nicotinic acetylcholine chemically distinguishable electrocytes appeared. From receptor, a-subunit); AE1 (anti-acidic keratin}; EA-53 lanti-a-ac- then on the electrocytes matured as these processes contin- tinin); CHI (anti-tropomyosin); 3A10 (anti-neurofilament-associ- ued at the leading edge of the blastema. ated protein). Following incubation in the primary antibody, sections were washed 3 × 5 mm in BSA/PBS, and incubated 60 rain m fluoreseem- conjugated secondary antibody (Fc'-specific anti-mouse, 1:100; Degenerating Electrocytes Cappell) to visualize primary antibody labeling. As a control, primary antibody was omitted, and sections were treated with only In electrocytes transected by the amputation, the cyto- the secondary antibody. In no ease was nonspecific labeling or plasm constricted, forming long (500-2000 #m), thin cells bright tissue autofluoresence observed. of much less overall diameter (<100 #m versus 300+ #m Images of labeled cells were recorded using a Nikon Diaphot for intact cells) than nontransected electrocytes m other epifluorescence microscope, a Cohu 4915 video camera, and a Colo- regions of the EO (Fig. 2). A concentric band of melanocytes rado Video frame store, interfaced to a Macintosh Quadra 800 run- and extracellular matrix material surrounded the collapsed ning NIH Image 1.47 software. Spinal cord transection. The electromotor neurons that inner- electrocyte. Like normal electrocytes, these cells were im- vate the EO are driven by neurons in the medulla. If the descending munoreactive for desmin, keratin, a-actinin, and acetylcho- axons of those neurons are severed, the electromotomeurons are line receptor (Fig. 2). In contrast to normal electrocytes (Pat- quiescent in this species (Bennett, 1971). To silence the EO, the terson and Zakon, 1993), the degenerating electrocytes were spinal cord was transected at the level of the pectoral fin in eight immunoreactive for myosin [Figs. 2A and 2B). Myosin label more fish. Fish were anesthetized in MS-222, and an incision was was diffuse and filled the cell with a meshwork of labeled made in the skin through the muscle and down to the vertebral filaments, a-Actinin labeling followed two patterns: some column. A scalpel was then used to chip through the vertebra. cells showed disorganized a-actinin labeling, whereas oth- Once the cord was exposed, fine forceps were used to tease it until ers displayed a striated pattern of a-actinin label (Fig. 2C). it was visibly sectioned. The wound was sutured and fish were For up to 10 days postamputation, the degenerating elec- revived and returned to their aquaria. To confirm the absence of an electrical organ discharge, electrocytes demarcated the transection site, although by this trodes were placed next to the fish after surgery, periodically while time, a regeneration blastema has extended 2-3 mm beyond they were in their aquaria, and lust prior to removal of the regenera- the cut.

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Day 0 Day 4 Day 6 Day 8

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Day 10 Day 12 Day 14 v

FIG. 1. Schematic diagram of electric organ regeneration. At Day 0, the EO is removed, leaving a stump, consistmg of intact (open ovals) and transected (half ovals) electrocyte cells. The small black ovals are the muscle fibers. On Day 4, transected electrocytes are degenerating (thin lines) and lie between mtact electrocytes and pockets of mesenchymal cells that form at the wound margin (shaded region). On Days 6-8, the mesenchymal cells begin to form a blastema that is visible externally. Signs of myogenesis begin at the border between the blasterna and the stump (small black dots adjacent to blastema). At Day 10 immunochemically idennfiable electrocytes (small open ovals) are first seen. On Day 12, the wave of myogenesis continues to move distally as the blastema extends farther. By Day 14, the distribution of regenerated muscle and electrocytes closely resembles that of intact EO. Arrows mdicatc the level of cross sections made at Day 12 in subsequent figures. All subsequent longitudinal sections are oriented with the blastema to the right as m this figure.

Myogenesis and the Expression of Sarcomeric desmin-positive cells are nascent electrocytes. This view is Proteins in the Blastema reinforced by examining sections 150 #m proximal in which The first indication of differentiation within the blastema there was clear segregation into muscle and electrocytes was the formation of small aggregations of mesenchymal (Fig. 4C). Large desmin-positive cells, 200 #m or more in cells at the most distal tip. These cell clusters did not ini- diameter were situated below clusters of skeletal muscle tially express any muscle-specific epitopes. Clusters located fibers. Segregation was not complete; sometimes small a few hundred micrometers more proximal, however, muscle fiber-like cells were interspersed within the layer strongly expressed desmin, which is an early marker of the of larger developing electrocytes (Fig. 4D, double arrows). muscle phenotype (Lazarides et al., 1982; Debus et al., 1983; These were confirmed to be smaller muscle fibers by follow- Danto and Fischman, 1984; Tokuyasu et al., 1984) (Fig. 3). ing them in serial sections. The complete pattern of myogenesis within the blastema Desmin was expressed in differentiating electrocytes can be seen by examining cross sections of a 12-day regener- throughout the blastema, and within all the electrocytes of ate made at closely spaced intervals and labeled with the the intact EO. This is not surprising since it is expressed desmin Ab. In the most distal regions, desmin immunoreac- in mature electrocytes (Patterson and Zakon, 1996). tivity appeared within a bilaterally paired group of ceils in Single electrocytes were large enough to be tracked the ventromedial blastema (Fig. 4A). Within a single sec- through many sections. By examining adjacent serial cross tion, each group of desmin-positive cells numbered between sections labeled with other Abs, we found that desmin- one and two dozen in the postenormost sections where they positive cells also expressed myosin, tropomyosin, and a- were first detected. At the periphery were small desmin- actinin. Within the peripherally located smaller muscle positive cells. These cells were less than 50 #m in cross fibers (Fig. 5, small arrow), a-actinin was found in well- section, and were loosely aggregated in clusters of 8-20 aligned Z-lines. In contrast, within nascent dectrocytes a- cells (Fig. 4B, double arrows). More centrally were larger actinin labeling was dimmer and not well-aligned; EA-53 desmin-positive cells; these cells were elliptical in cross labeled a disrupted network of striated myofibrils that filled section and were between 100 and 200 #m in diameter (as the electrocyte (Fig. 5, large arrow). This same pattern was measured along the long axis) (Fig. 4B, single arrow). observed with the myosin Ab MF20 (data not shown). Based on their size and position, we believe that the Often small brightly labeled muscle-like cells were seen smaller peripherally located desmin-positive cells become deep within the central section of the blastema, far from the skeletal muscle, and that the larger more centrally located ulnmate location of muscle (Fig. 5, double arrows). Small

FIG. 3. Expression of desmin (D76) immunoreactivity in brightly labeled clusters of blastemal cells in an 8-day regenerate was the first indication of differentiation. This image was purposely overex- posed to show the desmin-negative cells located m the more distal blastema. Longitudinal section with the tip of the blastema on the right. Scale bar, 100 #m.

muscle fibers with well-aligned bright ~-actinin striations could be seen in immediate proximity to electrocytes. In- side some electrocytes there were small regions where a- actinin and myosin labeling was intense, and striations were predominant. These may represent sites of recent myotube/electrocyte fusion. Interestingly, while myosin expression was evident in the developing electrocytes, tropomyosin expression was re- stricted to the muscles, even those in the core of the blastema (data not shown), suggesting that it is rapidly down- regulated once muscles convert to electrocytes (Fig. 6). A distractive histochemical feature of mature electrocytes of Sternopygus is keratin (Patterson and Zakon, 1996) so we determined when keratin is expressed during regeneration and whether it is coexpressed with muscle-specific proteins. AE1 anti-keratin staining was first detected within the middle of the blastema in large (200+ #m in length) elliptical electrocytes (Fig. 7). Electrocytes of this size still diffusely expressed cr-actinin and myosin, although no dis- cernible sarcomeric organization remained (Fig. 71. Newly generated muscle cells were also lightly labeled with AE1 Ab (Fig. 8). Myosin staining was less intense or absent in the larger more differentiated electrocytes (Fig. 8). In some 12- and 14- FIG. 2. Degenerating electrocytes near the wound margin tran- day fish, lightly staining myosin-positive electrocytes pos- siently express muscle proteins. (A) In a longitudinal section of the teriorally and myosin-negative electrocytes anteriorally EO stump 4 days after transection the anti-myosin antibody MF20 demarcate the site of the transection (Fig. 9). Since electro- labeled both muscle (M) and degenerating electrocytes (middle of cytes first appear on Day 8, myosin immunoreactivity re- figure], but did not label intact electroeytes (E). Under higher mag- mains for about a week after the sarcomeres are first ob- nification labeling with MF20 (B) or the anti-c~-actinin antibody served to disappear. At these times cr-actinin was localized EA-53 {C) sarcomeres were visible in the degenerating electrocytes mainly at the posterior membrane where synapses are 6 days after transection. Scale bars: 50/~m in A, 25 in B, and 50 made. ~m in C. The sequence of these changes in immunohistochemical

FIG. 4. A series of cross sections through a lateral portion of a 12-day regeneration blastema labeled with D76 anti-desmin (rotated by 90 ° so that lateral is up). (A) Approximately 200 gm from the tip of the blastema, several dozen cells that lie beneath the skin (S) were the first to express desmin. At 150 /~m proximal (B), this pool of desmin-positive cells had expanded, and consisted of small-diameter cells found principally at the periphery (double arrows) and larger-diameter cells found centrally (single arrow). At 150 #m more proximal (C), the mature pattern of muscle fiber bundles at the penphery (double arrows) and electroeytes (single arrow) centrally was more striking. (D) A higher magmfication of the section in B shows that segregation into layers of muscle or electrocyte was not complete; small- diameter fibers could be found eentrally (single arrow), sometimes adjacent to larger electroeytes (double arrows). Scale bars, 100 ~m in all panels.

profiles of these proteins and the times at which they are rain-positive cells that first appeared in the blastema dis- first observed in summarized in Fig. 12. played punctuate 88b anti-AChR labeling similar to that seen in normal muscle fiber NMJ (Fig. 11). More centrally Nerve Fibers and Acetylcholine Receptor the larger electrocytes showed thread-like aggregations at the cell periphery with mAb 88b (Fig. 11). Serial sections revealed that axonal ingrowth preceded the differentiation of the myotubes (Fig. 10). Alternate adjacent sections labeled with D76 or MF20 and 3AlO showed Effects of Spinal Cord Transection on Electrocyte that no cells in the region immediately surrounding these Differentiation in-growing axons expressed a myogenic phenotype. At 150 To ascertain the role of activity in the regeneration of the #m proximal, however, early myogenic events were ob- electric organ, high spinal transections were made at the served precisely in locations pioneered by axons (Fig. 10). time of EO amputation in another group of fish. Such tran- Interestingly, occasional clusters of myosin-positive cells sections cut the relay axons that project from the brain stem could be seen in the interior of the blastema. These were to the electromotor neurons, thereby silencing the electric always surrounded by nerve fibers. organ discharge (EOD). Recordings made with electrodes Acetylcholine receptor (AChR) was expressed during alongside the electric organ verified that the electric organ early differentiation of the muscle. Some of the small des- was not firing during this time. We then repeated the above

active, neurons grew into the blastema, where normal histogenesis of both electrocytes and muscle occurred. By Day 14, electrocytes and muscle fibers differentiated fully despite the lack of neural activity and no differences were observed in the time course or pattern of their reinnerva- tion.

DISCUSSION Degenerating Electrocytes At 4 days posttransection, degenerating electrocytes showed diffuse myosin expression. Later, anti-myosin and anti-~-actinin antibodies revealed striations. During this same time, the degenerating electrocytes continued to express "old" epitopes, such as desmin, keratin, and AChR. FIG. 5. Cross section from a 12-day regenerate labeled with anti- ~-actmin antibody. In muscle (small single arrow), a~actinin-la- These results raise the question: are electrocytes reverting beled sarcomeres were tightly aligned. Within electrocytes (large to a more muscle-like phenotype or have new myogenic arrow), the sarcomeres were not aligned, although striations were ceils infiltrated the transected cells and differentiated into still visible. Muscle fibers were oeeasionally observed in close prox- muscle? Our results cannot rule out either possibility but, imity to electrocytes (double arrows). Scale bar, 100 ~m. based on our earlier demonstration that bromodeoxyuri- dine-labeled satellite cells can be found within the degenerating electrocytes by at least 6 days after tail amputation (Patterson and Zakon, 1993), we favor the latter hypothesis. sectioning and immunostaining of the blastema during re- Degenerating electrocytes might function like the basal generation (Day 7, n = 2; Day 10, n = 3; Day 14, n = 3). lamina ghosts of skeletal muscle. When a muscle fiber de- Although they received no input and were, therefore, in- generates due to injury, its basal lamina acts as a scaffold

FIG. 6. Adjacent cross sections from a 14-day regenerate labeled with antibodies agamst myosin (A) and tropomyosin (B) show that while myosin is expressed in both large electrocytes (E) and small muscle fibers (M), tropomyosin is only observed in muscle cells. Scale bar, 1O0/~m.

FIG. 7. Maturing electrocytes from a 10-day regenerate coexpress muscle- and electrocyte-specific epitopes. (A) Newly formed keratm- pomtive electrocyte (arrow) in the vicinity of intact electrocytes. (B) The cell in A seen at higher magnification. In adjacent sections this electrocyte also labeled with MF20 anti-myosin (C), and EA-53 antl-~-actinin (D). Scale bar, 100 #m in all panels.

providing cellular cues to aid the fiber's regeneration. Myo- highly organized myofibrils with Z-lines in tight register, satellite cells proliferate within this basal lamina tube whereas the alignment of myofibrils is disrupted within the (Bischoff, 1974, 1986), and extracellular matrix molecules larger nascent electrocytes. remain its surface to guide new synaptic contacts to the The location of some muscle fibers deep within the core site of the previous neuromuscular junction (Sanes, 1983). of the blastema adjacent to electrocytes suggests that the Electrocytes and muscle are surrounded by a population of electrocytes might gain their large size from the fusion of myosatellite cells that proliferate to form new muscle and differentiated muscle ceils (Fig. 5, double arrows). While electrocytes. Initially, the satellite cells aggregate along the this hypothesis cannot be tested from immunocytochemi- length of, and within, the transectcd electrocytes filling the cal data, electron micrographs of regenerating EO show re- collagen shells of extracellular matrix material that sur- gions of cytoplasmic continuity between small groups of round the degenerating cells (Patterson and Zakon, 1993). muscle cells that can be interpreted as fusion (G. Unguez and Y. Lu, unpublished data). The disassembly of the contractile apparatus and dissolu- Electrocyte Differentiation within the Blastema tion of myofibrils has been reported in many cases of elec- and Sarcomeric Disorganization trocyte regeneration or development with ultrastruetural Both electrocytes and skeletal muscle arise from a pool methods (Szabo, 1960; Keynes, 1961; Baillet-Derbin, 1978~ of myogenic precursor ceils located in the regeneration blas- Patterson and Zakon, 1993). Immunoeytoehemistry, how- tema. Myogenesis, as detected by our musele-speeiflc cell ever, has allowed us to observe some new aspects of this markers, begins within a group of otherwise identical mes- process. Despite the disappearance of the myofibrillar appa- enchymal cells in the proximal ventromedial region of the ratus, the electrocytes do not cease production of all myofi- blastema, and moves posteriorally and dorsally as a wave brillar proteins and those that disappear do so at different of differentiation. At the leading edge of this wave and pre- times. Tropomyosin disappears rapidly, myosin is present ceding it are nerve fibers, followed closely by mononucleate for at least a week after the dissolution of the sarcomeres, cells that express a desmin epitope, presumably myoblasts. while desmin and a-actinin continue to be produced in ma- The myoblasts fuse to form striated myotubes which then ture electrocytes (Patterson and Zakon, 1996). Whether the segregate into peripherally located cells that continue to difference in the timing of disappearance of myosin and express a muscle phenotype, and centrally located cells that tropomyosin is due to differential transcriptional control or transdifferentiate into electrocytes. Although at the earliest protein degradation is unknown. Nevertheless, the fact that times both cell types express markers common to muscle mature electrocytes lack myosin and tropomyosin while (desmin, myosin, tropomyosin, o~-actinin, and AChR), the desmin is abundant suggests that these myofibrillar pro- two cell types can be distinguished by their size and pattern teins are independently regulated during the conversion of striations. Muscle fibers are small (50 #m) and contain from muscle to electrocyte much as they are during initial

Copyright © 1997 by Academic Press All rights of reproduction m any form reserved. 122 Patterson and Zakon development of muscle {Wang et al., 1988; Page et aI., 1992; Lin et al., 1994; Rhee et aI., 1994). It would be interesting to learn the fates of other myofibrillar proteins, such as ~-actin, as well as those that orga- nize the myofibrillar proteins into sarcomeres such as zeug- matin and titin and to determine what initiates disruption of the sarcomeres and myofibrillar breakdown in electrocytes. This process might provide clues to the normal events involved in the establishment of sarcomeres initially. Recent work with cultured Xenopus myocytes shows that calcium transients from internal sources are necessary for the elaboration of sarcomeres but not for the maturation of membrane ionic currents or AChRs (Ferrari et al., 1996).

FIG. 9. At the site of the transection, sometimes a boundary was visible between intact myosin-negative electrocytes within the original EO (left side of figure), and newly regenerated myosin- positive electrocytes (right side of figure) that formed within the blastema. Fourteen-day regenerate, longitudinal section. Scale bar, 100 #m.

This is in agreement with the disruptions of sarcomeric organization that occur in mice and chickens with muta- tions of the ryanodine receptor [Airey et al., 1993; Takekura et aI., 1995). These studies suggest that the ryanodine receptor may be an important molecule in the regulation of the electrocyte phenotype. Mature electrocytes do not possess any sarcoplasmic reticulum thus they would be unlikely to have ryanodine receptors.

Transient Coexpression of Muscle and Electrocyte Phenotypes As Sternopygus electrocytes begin to form and while they are still expressing myosin, they also express an isoform of keratin that is characteristic of mature electrocytes. As electrocytes differentiate, therefore, they transiently coexpress components of both muscle and electrocyte phenotypes. A similar example of transdifferentiation of muscle tissue occurs during development of the mammalian esoph- agus in which smooth muscle converts to striated muscle after expressing a mixed phenotype (Papapoutian et al., 1995). In addition to keratin, androgen and estrogen receptors have been localized in mature electrocytes but not in adjacent muscle fibers (Gustavson et al., 1994; Dunlap et aI., 1996). We have not yet determined how these proteins are regulated during regeneration of the muscles or electro- FIG. 8. Adjacent cross sections from the blastema/stump bound- cytes. Thus, electroeytes are more than just partially sup- ary from a 12-day regenerate labeled with AE1 anti-keratin (A) or pressed muscles, they express proteins that are not detected MF20 anti-myosin (B). All electrocytes were keratin-positive, some in differentiated muscle. of the smaller electrocytes were still myosin-positive (large arrow in A and B), whereas other, usually larger, electrocytes were myo- Role of Innervation sin-negative (double arrows in A and B). Fascicles of muscle fibers are indmated by asterisks. Note weak expression of keratin in some In mammalian muscle, fiber type and associated changes muscle fibers. Scale bar, 100 ~m. in gene expression are plastic; numerous manipulations

FIG. 10. Cross sections of a single 12-day regeneration blastema labeled with 3A10 neurofilament antibody. At the most distal pornon of the blastema (A), only a few axons were present (arrow). No other differentiated cell type could be identified at this level. Progressing proximally in 150-#m increments (B, C, D), more axons were observed within the blastema just beneath the skin (S). (E) Approximately 600 #m proximal to the section in A, evidence of cellular differentiation was first evident (visible condensations of blastemal ceils, at arrows). No differentiation was observed in noninnervated regions of the blastema. At the most proximal level examined (F), newly formed electrocytes (single arrow) were developing in the central regions of the blastema within range of innervation. Scale bar, 100 #m.

Muscles and electrocytes are innervated by different pop- ulations of motorneurons with distinct morphologies and electrical behavior. Large-diameter electromotor neurons, which are present in the central region of the spinal cord, are driven continuously at rates of 100-200 Hz in this species (Zakon, 1996). Smaller somatomotor neurons innervate muscles in the tail that we presume control tail position; these must fire irregularly. One attractive hypothesis is that the conversion of muscle cells to electrocytes is induced by the regular firing pattern of electromotor neurons, while muscle cells contacted by normal somatic motor neurons remain unchanged. Al- though we do not know when electrical stimulation of the electrocytes commences, myofibers are innervated (although the identity of the axons cannot be determined), and AChRs are present during the initial stages of myotube formation making this a plausible hypothesis. However, the FIG. 11. Acetylcholine receptors were expressed on the surface of nascent electrocytes. Cross sections from a 12-day regenerate. fact that EO develops normally in spinally transected fish Scale bar, 100/~m. whose electromotorneurons are silent argues against any role for repetitive neural activity in electromotorneurons causing phenotype conversion. In this sense conversion of muscle to eleetrocyte is differ- have shown how the pattern of electrical stimulation can ent from conversion of one fiber type of mammalian muscle alter the cell phenotype. Cross-innervation studies have to another. It would be interesting to examine whether fast demonstrated the ability of nerve activity to change the and slow muscles in fish (Karasinski, 1993) are interconvert- expression of specific myofibrillar genes within muscle ible and, if so, whether this is based on electrical activity (Salmons and Sreter, 1976; Hughes et al., 1993). If a slow as in mammals or on a non-activity-dependent process. muscle fiber is reinnervated by a fast motor neuron, levels Since neural activity does not seem to direct the fate of of MHCf~st rise, whereas levels of MHCslo,~ diminish. Expres- these cells, we would conclude that the final cell fate is sion of myogenic transactivating factors such as MyoD and being determined by one of three possible mechanisms: (1) myogenin are differentially expressed in muscles of differ- through an interaction with the nerve that operates inde- ent fibers types, and are under neural regulation by electri- pendently of nerve activity (i.e., release of specific factors cal stimulation (Buonanno et aI., 1992; Hughes et al., 1993; by the nerve), (2) by distinct classes of satellite cells that Michel et al., 1996). generate either muscle or electrocytes, or (3) the developing

differentiation fusion dissolution as of of muscle myotubes (?) sarcomeres l desmin

ACh receptor

myosin

tropomyosin

~-actinin low levels at post-synaptic membranes

Keratin

8 days 10 days 12 days 14 days

FIG. 12. Schemanc illustration of regulation of the markers observed in the electrocytes. Levels are given by the width of the bar.

Copyright © 1997 by Academic Press. All rights of reproduction m any form reserved. Transdifferentiation of Muscle to Electric Organ 125 cell's location within some type of morphogenetic gradient. MyoD: Myogenin mRNAs m fast and slow adult skeletal muscle Future work will be aimed at testing these hypotheses. is controlled by innervation and hormones. Development 118, 1137-1147. Karasinski, J. (1993). Diversity of native myosin and myosin heavy chain in fish skeletal muscles. Comp Blochem. Physiol. B 106, ACKNOWLEDGMENTS 1041-1047. Keynes, K. D. (1961). The development of the electric organ in Elec- The authors thank Dr. Graciela Unguez for providing the data trophorus electricus. In "Bioelectrogenesis" (C. Chagas and A. in Fig. 6; Susan Gustavson for animal maintenance; Juliette Erikson Paes de Carvalho, Eds.), pp. 14-18. Elsevier, Amsterdam. for assistance m the laboratory; Janet Young, Gwen Gage, and Kris- Larochelle, W. J., Witzemann, V., Fiddler, W., and Froehner, S. 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