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This paper presents a novel wireless underwater data acquisition system for sensing electric organ discharge (EOD) signals generated from the weakly electric fish S. macrurus . Variation in frequency and amplitude of the EOD signals are of interest by biologists to study behavioral and environmental effects on electric organ cells. In order to record the EOD signals, a miniature wearable wireless sensing system is designed for the target fish. The system consists of a custom-designed integrated analog front-end (AFE), an ATmega328p microcontroller unit and a wireless transmitter (TX), as well as a battery with a power management module. In order to save TX power, the wireless sensor only transmits the calculated frequency and amplitude information of the EOD signal based on a proposed gain-feedback control method. A receiver attached to a host computer receives the wirelessly transmitted EOD data to perform further analysis. A wearable waterproof backpack for the fish is designed to house the wireless sensor and battery. The overall system has been successfully tested in a clinical experiment with the weakly electric fish. The AFE integrated circuit, which includes a novel rail-to-rail dynamic comparator, is fabricated in a 0.18- $\mu \text{m}$ CMOS process. The total area of the core circuit is 1.0 mm2. The LNPA achieved a noise efficiency factor of 1.8. The AFE power consumption is 2.2 $\mu \text{W}$ from a single +1.8 V supply, whereas the overall system power consumption is 15 mW. The proposed gain-feedback control structure for frequency and amplitude measurement saves the transmitter data rate by 3,756 times compared with sending the raw data.

Scientific societies aiming to foster inclusion of scientists from underrepresented (UR) backgrounds among their membership often delegate primary responsibility for this goal to a diversity-focused committee. The National Science Foundation has funded the creation of the Alliance to Catalyze Change for Equity in STEM Success (ACCESS), a meta-organization bringing together representatives from several such STEM society committees to serve as a hub for a growing Community of Practice. Its goal is to coordinate efforts to advance inclusive practices by sharing experiences and making synergistic discoveries about what works. ACCESS has analyzed the approaches by which member societies have sought to ensure inclusivity through selection of annual meeting speakers. Not only do we discuss how inclusive speaker selection fosters better scientific environments for all, but also identify challenges and promising practices for societies striving to maximize inclusivity of speakers in their scientific programming.

Electrical activity is an important regulator of cellular function and gene expression in electrically excitable cell types. In the weakly electric teleost fish Sternopygus macrurus, electrocytes, i.e., the current-producing cells of the electric organ, derive from a striated muscle lineage. Mature electrocytes are larger than muscle fibers, do not contain sarcomeres, and are driven continuously at frequencies higher than those exerted on muscle cells. Previous work showed that the removal of electrical activity by spinal cord transection (ST) for two and five weeks led to an upregulation of some sarcomeric proteins and a decrease in electrocyte size. To test whether changes in gene transcription preceded these phenotypic changes, we determined the sensitivity of electrocyte gene expression to electrical inactivity periods of two and five days after ST. Whole tissue gene expression profiles using deep RNA sequencing showed minimal alterations in the levels of myogenic transcription factor and sarcomeric transcripts after either ST period. Moreover, while analysis of differentially expressed genes showed a transient upregulation of genes associated with proteolytic mechanisms at two days and an increase in mRNA levels of cytoskeletal genes at five days after electrical silencing, electrocyte size was not affected. Electrical inactivity also resulted in the downregulation of genes that were classified into enriched clusters associated with functions of axon migration and synapse structure. Overall, these data demonstrate that unlike tissues in the myogenic lineage in other vertebrate species, regulation of gene transcription and cell size in the muscle-like electrocytes of S. macrurus is highly insensitive to short-term electrical inactivity. Moreover, together with data obtained from control and long-term ST studies, the present data suggest that neural input might influence post-transcriptional processes to affect the mature electrocyte phenotype.

Skeletal muscle is distinguished from other tissues on the basis of its shape, biochemistry, and physiological function. Based on mammalian studies, fiber size, fiber types, and gene expression profiles are regulated, in part, by the electrical activity exerted by the nervous system. To address whether similar adaptations to changes in electrical activity in skeletal muscle occur in teleosts, we studied these phenotypic properties of ventral muscle in the electric fish Sternopygus macrurus following 2 and 5 days of electrical inactivation by spinal transection. Our data show that morphological and biochemical properties of skeletal muscle remained largely unchanged after these treatments. Specifically, the distribution of type I and type II muscle fibers and the cross-sectional areas of these fiber types observed in control fish remained unaltered after each spinal transection survival period. This response to electrical inactivation was generally reflected at the transcript level in real-time PCR and RNA-seq data by showing little effect on the transcript levels of genes associated with muscle fiber type differentiation and plasticity, the sarcomere complex, and pathways implicated in the regulation of muscle fiber size. Data from this first study characterizing the acute influence of neural activity on muscle mass and sarcomere gene expression in a teleost are discussed in the context of comparative studies in mammalian model systems and vertebrate species from different lineages.

Community-based participatory research can help reduce health disparities among underserved populations. The New Mexico State University - Fred Hutchinson Cancer Research Center Partnership for the Advancement of Cancer Research (NMSU – Fred Hutch PACR) aims to reduce cancer health disparities in part through the establishment of community-academic partnerships. The Outreach Core of the PACR has supported a small grants program since 2015 to award funds to competitive community-academic partnership teams addressing regional cancer or health disparities issues through health education. Each year, the Outreach Core staff presents a grant-writing workshop to assist community groups in the development of competitive proposals and to identify academic partners at NMSU. Participants at the grant-writing workshop submit proposals, which are then reviewed by two NMSU faculty researchers. All participants, including the awardees, receive the reviewers’ feedback on their proposal. Over the past four cycles, the grant-writing workshop has trained 60 community members. The top two proposals are funded each year. To date, eight different proposals have been funded, six at $7500 each and two at $5,000 each. All of the funded projects undergo an IRB review at NMSU before the start of the research project. The NMSU academic partners are involved with the community groups through the design, submission of Human Subjects protocol to the IRB, and through data analysis and reporting. Examples of small grant proposals will be presented, including a discussion of how their partnerships worked. This interaction between the community groups and research academics increases the relevance of academic research by assisting the community groups in evaluating their novel educational interventions through robust research projects. Furthermore, these small grant partnerships may serve to provide pilot data for more extensive efforts funded by other agencies. Citation Format: Karoline Sondgeroth, Rebecca Palacios, Graciela A Unguez, Mary O'Connell, Helena B Loest, Kaitlin Englund. Community-academic partners working in a small grants program [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B047.

Only one way to make an electric organ? Electric fish have independently evolved electric organs that help them to communicate, navigate, hunt, and defend themselves. Gallant et al. analyzed the genome of the electric eel and the genes expressed in two other distantly related electric fish. The same genes were recruited within the different species to make evolutionarily new structures that function similarly. Science , this issue p. 1522

SUMMARYIn most groups of electric fish, the current-producing cells of electric organs (EOs) derive from striated muscle fibers but retain some phenotypic characteristics of their precursor muscle cells. Given the role of the MyoD family of myogenic regulatory factors (MRFs) in the transcriptional activation of the muscle program in vertebrates, we examined their expression in the electrocytes of the gymnotiform Sternopygus macrurus. We estimated the number of MRF genes in the S. macrurus genome and our Southern blot analyses revealed a single MyoD, myogenin, myf5 and MRF4 gene. Quantitative RT-PCR showed that muscle and EO transcribe all MRF genes. With the exception of MyoD, the endogenous levels of myogenin, myf5 and MRF4 transcripts in electrocytes were greater than those detected in muscle fibers. These data indicate that MRF expression levels are not sufficient to predict the level to which the muscle program is manifested. Qualitative expression analysis of MRF co-regulators MEF2C, Id1 and Id2 also revealed these genes not to be unique to either muscle or EO, and detected similar expression patterns in the two tissues. Therefore, the partial muscle program of the EO is not associated with a partial expression of MRFs or with apparent distinct levels of some MRF co-factors. In addition, electrical inactivation by spinal cord transection(ST) resulted in the up-regulation of some muscle proteins in electrocytes without an accompanying increase in MRF transcript levels or notable changes in the co-factors MEF2C, Id1 and Id2. These findings suggest that the neural regulation of the skeletal muscle program via MRFs in S. macrurus might differ from that of their mammalian counterparts. Together, these data further our understanding of the molecular processes involved in the plasticity of the vertebrate skeletal muscle program that brings about the muscle-like phenotype of the non-contractile electrogenic cells in S. macrurus.

*Department of Biology 42451, University of Louisiana at Lafayette, Lafayette, LA 70504, U.S.A., †Section of Neurobiology, School of Biological Sciences, University of Texas at Austin, P. O. Box 7608, Austin, TX 78713-7608, U.S.A., ‡Department of Biological Sciences, Florida International University, Miami, FL 33199, U.S.A., §Department of Biology, Foster Hall 3AF, New Mexico State University, Las Cruces, NM 88003, U.S.A., kDepartment of Arts and Sciences, University of Phoenix, Lafayette, LA 70504, U.S.A. and {Biotechnology Center, 425 Henry Mall, University of Wisconsin, Madison, WI 53706, U.S.A.

In most electric fish species, the electric organ (EO) derives from striated muscle cells that suppress many muscle properties. In the gymnotiformSternopygus macrurus, mature electrocytes, the current-producing cells of the EO, do not contain sarcomeres, yet they continue to make some cytoskeletal and sarcomeric proteins and the muscle transcription factors (MTFs) that induce their expression. In order to more comprehensively examine the transcriptional regulation of genes associated with the formation and maintenance of the contractile sarcomere complex, results from expression analysis using qRT-PCR were informed by deep RNA sequencing of transcriptomes and miRNA compositions of muscle and EO tissues from adultS. macrurus. Our data show that: (1) components associated with the homeostasis of the sarcomere and sarcomere-sarcolemma linkage were transcribed in EO at levels similar to those in muscle; (2) MTF families associated with activation of the skeletal muscle program were not differentially expressed between these tissues; and (3) a set of microRNAs that are implicated in regulation of the muscle phenotype are enriched in EO. These data support the development of a unique and highly specialized non-contractile electrogenic cell that emerges from a striated phenotype and further differentiates with little modification in its transcript composition. This comprehensive analysis of parallel mRNA and miRNA profiles is not only a foundation for functional studies aimed at identifying mechanisms underlying the transcription-independent myogenic program inS. macrurusEO, but also has important implications to many vertebrate cell types that independently activate or suppress specific features of the skeletal muscle program.

The neural mechanisms underlying changes in gene expression in the interconversion between skeletal muscle and the non-contractile electrogenic cells of the electric organ in electric fishes require several days to be manifested. It is extremely challenging to study these non-immediate forms of plasticity in reduced preparations in cell culture due to the time requirements. To address this experimental obstacle we developed a 3D-printed wearable backpack that allows chronic electrical stimulation of aquatic teleost fish. The backpack holds a biphasic simulator using a full H-bridge driver structure. Stimulation amplitude is adjusted with a current source controlled by a micro potentiometer whereas the stimulation waveform is reconfigurable through a micro-controller. A 3.7 V Lithium Ion Polymer battery powers the entire circuit. This backpack system will allow underwater chronic stimulation experiments aimed to study the role that neuronal input exerts on cell phenotypes in a vertebrate species with high tissue regeneration and cell trans-differentiation capabilities.

Background With its unique ability to produce high-voltage electric discharges in excess of 600 volts, the South American strong voltage electric eel (Electrophorus electricus) has played an important role in the history of science. Remarkably little is understood about the molecular nature of its electric organs. Results We present an in-depth analysis of the genome of E. electricus, including the transcriptomes of eight mature tissues: brain, spinal cord, kidney, heart, skeletal muscle, Sachs’ electric organ, main electric organ, and Hunter’s electric organ. A gene set enrichment analysis based on gene ontology reveals enriched functions in all three electric organs related to transmembrane transport, androgen binding, and signaling. This study also represents the first analysis of miRNA in electric fish. It identified a number of miRNAs displaying electric organ-specific expression patterns, including one novel miRNA highly over-expressed in all three electric organs of E. electricus. All three electric organ tissues also express three conserved miRNAs that have been reported to inhibit muscle development in mammals, suggesting that miRNA-dependent regulation of gene expression might play an important role in specifying an electric organ identity from its muscle precursor. These miRNA data were supported using another complete miRNA profile from muscle and electric organ tissues of a second gymnotiform species. Conclusions Our work on the E. electricus genome and eight tissue-specific gene expression profiles will greatly facilitate future research on determining the coding and regulatory sequences that specify the function, development, and evolution of electric organs. Moreover, these data and future studies will be informed by the first comprehensive analysis of miRNA expression in an electric fish presented here. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1288-8) contains supplementary material, which is available to authorized users.

The electromotor and electrosensory systems of the weakly electric fish Apteronotus leptorhynchus are model systems for studying mechanisms of high-frequency motor pattern generation and sensory processing. Voltage-dependent ionic currents, including low-threshold potassium currents, influence excitability of neurons in these circuits and thereby regulate motor output and sensory filtering. Although Kv1-like potassium channels are likely to carry low-threshold potassium currents in electromotor and electrosensory neurons, the distribution of Kv1 α subunits in A. leptorhynchus is unknown. In this study, we used immunohistochemistry with six different antibodies raised against specific mammalian Kv1 α subunits (Kv1.1–Kv1.6) to characterize the distribution of Kv1-like channels in electromotor and electrosensory structures. Each Kv1 antibody labeled a distinct subset of neurons, fibers, and/or dendrites in electromotor and electrosensory nuclei. Kv1-like immunoreactivity in the electrosensory lateral line lobe (ELL) and pacemaker nucleus are particularly relevant in light of previous studies suggesting that potassium currents carried by Kv1 channels regulate neuronal excitability in these regions. Immunoreactivity of pyramidal cells in the ELL with several Kv1 antibodies is consistent with Kv1 channels carrying low-threshold outward currents that regulate spike waveform in these cells (Fernandez et al., J Neurosci 2005;25:363–371). Similarly, Kv1-like immunoreactivity in the pacemaker nucleus is consistent with a role of Kv1 channels in spontaneous high-frequency firing in pacemaker neurons. Robust Kv1-like immunoreactivity in several other structures, including the dorsal torus semicircularis, tuberous electroreceptors, and the electric organ, indicates that Kv1 channels are broadly expressed and are likely to contribute significantly to generating the electric organ discharge and processing electrosensory inputs. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006

After axonal injury on postnatal day 14 (P14), but not P21, motoneurons in the spinal nucleus of the bulbocavernosus (SNB) do not display their normal response to circulating testosterone levels. This could result from a permanent disruption of communication between motoneurons and their testosterone-sensitive target muscles. We assessed the extent of reinnervation of one of these target muscles, the levator ani (LA) muscle, 5 months after the pudendal nerve was cut either on P14 or P21. The number of motoneurons innervating the LA in control and nerve cut animals was determined using retrograde labeling procedures. Functional recovery of the LA muscle was determined via the testing of its in situ contractile properties. Compared to control muscles, reinnervated LA muscles were smaller, had fewer muscle fibers, generated a lower maximum tetanic tension, and were more fatigable. In spite of the fact that fewer motoneurons reinnervated the LA muscle after nerve cut on P14 than on P21, there were no differences in the weight or contractile properties of the LA muscle between these two groups. These data suggest that motoneurons that survived injury on P14 innervated more muscle fibers than normal and exhibited a similar ability to functionally reinnervate the target muscle as those motoneurons that survived injury on P21. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005

The expression of myosin heavy chain (MHC) isoforms leading to adult fiber phenotypes in the tibialis anterior (TA) and soleus muscles of the cat were investigated from embryonic day 35 to 1 year after birth. Electrophoresis and immunoblotting of myofibrils demonstrated the expression of 5 different MHC isoforms, i.e. I, IIa, IIx, embryonic, and neonatal, during development. Based on electrophoresis, the adult-like MHC composition of the soleus and TA were not observed until postnatal day 40 (P40) and 120 (P120), respectively. In contrast, immunohistochemical analyses revealed that the adult-like fiber phenotype composition was attained much later (P120) in the soleus. The existence of multiple MHC isoforms in individual fibers suggested that transitions occurred until P120 in both muscles. Adult type I fibers were first observed at P1. Adult IIA fibers were first observed at P30 in the TA and P40 in the soleus. IIX fibers were not identified until P40 in the TA. The transition to the predominantly slow phenotype of the soleus involved a gradual loss of embryonic and fast isoforms accompanied by an accumulation of slow MHC. In contrast, the expression of slow and fast MHC in the fast TA muscle was relatively unchanged throughout development. These results show that the establishment of a given MHC-based fiber phenotype varies significantly between slow and fast muscles in the kitten.

In most groups of electric fish, the electric organ (EO) derives from striated muscle cells that suppress many muscle phenotypic properties. This phenotypic conversion is recapitulated during regeneration of the tail in the weakly electric fish Sternopygus macrurus. Mature electrocytes, the cells of the electric organ, are considerably larger than the muscle fibers from which they derive, and it is not known whether this is a result of muscle fiber hypertrophy and/or fiber fusion. In this study, electron micrographs revealed fusion of differentiated muscle fibers during the formation of electrocytes. There was no evidence of hypertrophy of muscle fibers during their phenotypic conversion. Furthermore, although fish possess distinct muscle phenotypes, the extent to which each fiber population contributes to the formation of the EO has not been determined. By using myosin ATPase histochemistry and anti-myosin heavy chain (MHC) monoclonal antibodies (mAbs), different fiber types were identified in fascicles of muscle in the adult tail. Mature electrocytes were not stained by the ATPase reaction, nor were they labeled by any of the anti-MHC mAbs. In contrast, mature muscle fibers exhibited four staining patterns. The four fiber types were spatially arranged in distinct compartments with little intermixing; peripherally were two populations of type I fibers with small cross-sectional areas, whereas more centrally were two populations of type II fibers with larger cross-sectional areas. In 2- and 3-week regenerating blastema, three fiber types were clearly discerned. Most (. 95%) early-forming electrocytes had an MHC phenotype similar to that of type II fibers. In contrast, fusion among type I fibers was rare. Together, ultrastructural and immunohistochemical analyses revealed that the fusion of muscle fibers gives rise to electrocytes and that this fusion occurs primarily among the population of type II fibers in

SummaryBiology is replete with examples of regeneration, the process that allows animals 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 fishes 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 South America (Teleostei: Gymnotiformes). 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 electric fish Sternopygus macrurus (long-tailed knifefish) with a focus on regeneration of skeletal muscle and the muscle-derived electric organ. 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 animal 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.

The percent and distribution patterns of three immunohistochemically identified fiber types within the anterior compartment of the cat tibialis anterior were determined 6 months after denervation and self-reinnervation. After self-reinnervation, mean frequencies of slow (9%) and fast (91%) fibers were similar to those in control (12% and 88%, respectively) muscles. However, a lower proportion of fast-1 (26%) and a higher proportion of fast-2 (65%) fibers were observed in self-reinnervated than control (32% and 56%) muscles. Quantitation of adjacencies between fibers of similar myosin heavy chain (MHC) phenotype, a measure of type grouping, revealed that the frequencies of two slow or two fast-1 fibers being adjacent in self-reinnervated muscles were similar to control. In contrast, the frequency of fast-2/fast-2 fiber adjacencies found in self-reinnervated muscles (45%) was significantly higher than in control muscles (37%). In both groups, the frequency of adjacencies between slow, fast-1, or fast-2 fibers was largely attributable to the number of each fiber type present. These data show that the incidence of grouping within each fiber type present was not altered after 6 months of self-reinnervation. Minimal changes in the spatial distribution of fiber types following self-reinnervation in adults suggests a limited degree of conversion of muscle fibers to a MHC phenotype matching the motoneuron characteristics.

The ability to regenerate tissues is shared across many metazoan taxa, yet the type and extent to which multiple cellular mechanisms come into play can differ across species. For example, urodele amphibians can completely regenerate all lost tissues, including skeletal muscles after limb amputation. This remarkable ability of urodeles to restore entire limbs has been largely linked to a dedifferentiation-dependent mechanism of regeneration. However, whether cell dedifferentiation is the fundamental factor that triggers a robust regeneration capacity, and whether the loss or inhibition of this process explains the limited regeneration potential in other vertebrates is not known. Here, we studied the cellular mechanisms underlying the repetitive regeneration of myogenic tissues in the electric fish S. macrurus. Our in vivo microinjection studies of high molecular weight cell lineage tracers into single identified adult myogenic cells (muscle or noncontractile muscle-derived electrocytes) revealed no fragmentation or cellularization proximal to the amputation plane. In contrast, ultrastructural and immunolabeling studies verified the presence of myogenic stem cells that express the satellite cell marker Pax7 in mature muscle fibers and electrocytes of S. macrurus. These data provide the first example of Pax-7 positive muscle stem cells localized within a non-contractile electrogenic tissue. Moreover, upon amputation, Pax-7 positive cells underwent a robust replication and were detected exclusively in regions that give rise to myogenic cells and dorsal spinal cord components revealing a regeneration process in S. macrurus that is dependent on the activation of myogenic stem cells for the renewal of both skeletal muscle and the muscle-derived electric organ. These data are consistent with the emergent concept in vertebrate regeneration that different tissues provide a distinct progenitor cell population to the regeneration blastema, and these progenitor cells subsequently restore the original tissue.

1. The mechanical, morphological and biochemical properties of single motor units from the anterior compartment of the tibialis anterior muscle in adult cats were studied six months after the nerve branches to that compartment were cut and resutured in close proximity to the muscle. 2. In these self-reinnervated muscles, the maximum tetanic tensions were lower in slow than fast units, a relationship similar to that observed among motor units from control adult muscles. The maximum tetanic tensions produced by the fast units were larger than those produced by the same motor unit types in control muscles. Direct counts of muscle fibres belonging to a motor unit showed that factors controlling the number of muscle fibres innervated by a motoneurone type persist during the reinnervation process in that fast motoneurones reinnervated more muscle fibres than slow motoneurones. Thus, the number of muscle fibres reinnervated by a motoneurone principally accounted for the difference in the maximum tension outputs among motor unit types, a relationship similar to that observed in control tibialis anterior muscles. 3. Monoclonal antibodies for specific myosin heavy chains were used to differentiate fibre types. By this criterion, motor units from control muscles were found to contain a homogeneous fibre type composition. In contrast, a heterogeneous, yet markedly biased, fibre type composition was observed in each unit analysed from self-reinnervated muscles. 4. Although not all of the muscle fibres of a motor unit developed the same type-associated parameters after reinnervation, the relationships among myosin heavy chain profile, succinate dehydrogenase activity and the fibre size were similar in fibres of control and self-reinnervated muscles. 5. The processes which dictate both motor unit size and the matching between motoneurone and muscle fibre type during the reinnervation process must be interdependent and result from a hierarchy of decisions which reflects their relative importance. The mechanisms responsible for these two processes may be a combination of: (1) selective innervation which may or may not incorporate a pruning process if multiple synaptic connections are initially formed and/or (2) conversion of enough fibres of a motor unit to form a predominant type.

The spatial distribution of fibers belonging to a single motor unit was analyzed in 10 motor units from the tibialis anterior of the cat 6 months after denervation and self-reinnervation of the anterior (superficial) compartment of the muscle. After self-reinnervation, the distribution patterns of the fibers in the fast motor units were significantly different than control, whereas the fiber distribution in the slow unit was similar to control. Reinnervated fast units had a significant increase in the number of adjacencies among motor unit fibers, and there were often distinct “clusters or groups” of fibers distributed within the motor unit territory. Clustering or grouping of fibers was evident within the motor unit, even though fiber type grouping was not evident within the muscle. The differences in distribution patterns between control and reinnervated units may be related to variations in the branching pattern of axons during reinnervation compared to the process that occurs during development. © 1993 John Wiley & Sons, Inc.

The current-producing cells of the electric organ (EO), i.e., electrocytes, in Sternopygus macrurus derive from skeletal muscle fibers. Mature electrocytes are not contractile but they do retain some muscle proteins, are multinucleated, and receive cholinergic innervation. Electrocytes express the myogenic regulatory factors (MRFs) MyoD, myogenin, Myf5 and MRF4 despite their incomplete muscle phenotype. Although S. macrurus MRFs share functional domains that are highly conserved and their expression is confined to the myogenic lineage, their capability to induce the muscle phenotype has not been determined. To test the functional conservation of S. macrurus MRFs to transcriptionally activate skeletal muscle gene expression and induce the myogenic program, we transiently over-expressed S. macrurus MyoD (SmMyoD) and myogenin (SmMyoG) in mouse C3H/10T1/2 and NIH3T3 embryonic cells. RT-PCR and immunolabeling studies showed that SmMyoD and SmMyoG efficiently can convert these two cell lines into multinucleated myotubes that expressed differentiated muscle markers. The levels of myogenic induction by SmMyoD and SmMyoG were comparable to those obtained with mouse MRF homologs. Furthermore, SmMyoD and SmMyoG proteins were able to induce mouse MyoD and myogenin in C3H/10T1/2 cells. We conclude that S. macrurus MRFs are functionally conserved as they can transcriptionally activate skeletal muscle gene expression and induce the myogenic program in mammalian non-muscle cells. Hence, these data suggest that the partial muscle phenotype of electrocytes is not likely due to differences in the MRF-dependent transcriptional program between skeletal muscle and electric organ.

Tissue regeneration through stem cell activation and/or cell dedifferentiation is widely distributed across the animal kingdom. By comparison, regeneration in mammals is poor and this may reflect a limited dedifferentiation potential of mature cells. Because mammalian myotubes can dedifferentiate in the presence of newt blastema extract, the present study tested the dedifferentiation induction capability of the blastema from the teleost Sternopygus macrurus (SmBE). Our in vitro data showed that SmBE did not induce cell cycle reentry of myonuclei in myotubes. Instead, SmBE caused myotubes to detach and time-lapse imaging analyses characterized the cellular events before their detachment. Furthermore, SmBE enhanced myoblast proliferation and reversibly inhibited their differentiation. These data suggest the presence of protein factors in SmBE that regulate mammalian muscle physiology and differentiation, but do not support the conservation of a dedifferentiation induction capability by the blastema of S. macrurus.

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The MyoD family of basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs) are tran- scriptional activators of skeletal muscle gene expression and are pivotal in inducing the full myogenic program. The expression of these factors after muscle differentiation is complete and the mechanism by which they modulate (or maintain) the muscle phenotype is less well under- stood. The myogenically derived electric organ (EO) of the electric fish Sternopygus macrurus is an excellent model to address this question. The electrocytes, i.e., the electrogenic cells of the EO, are not contractile but they do retain some muscle proteins. In order to examine the molecular regulatory pathways that control the muscle-to- electrocyte cell conversion, we have cloned the MyoD and myogenin cDNAs from S. macrurus. Clustal-based alignments showed that the functional domains observed in mammalian MyoD and myogenin are highly conserved in these MRF homologs. Expression analyses revealed that mature electrocytes, which retain the muscle proteins dystrophin, desmin, acetylcholine receptors (AChRs), α- actin, and α-actinin, also transcribe the MyoD and myogenin genes. RT-PCR studies confirmed that expres- sion of these MRFs is confined to the myogenic lineage. Surprisingly, the levels of MyoD and myogenin transcripts in skeletal muscle and EO could not be used to predict the level to which a cell manifests the muscle program. We conclude that expression of multiple MRFs is not sufficient to induce non-contractile cells to fully express the skeletal muscle program. These data also suggest that the MRF transcriptional program in S. macrurus may be distinct from MRF-dependent myogenesis in other verte- brate systems.

The cells of the electric organ, called electrocytes, of the weakly electric fish Sternopygus macrurus derive from the fusion of mature fast muscle fibers that subsequently disassemble and downregulate their sarcomeric components. Previously, we showed a reversal of the differentiated state of electrocytes to that of their muscle fiber precursors when neural input is eliminated. The dependence of the mature electrocyte phenotype on neural input led us to test the hypothesis that innervation is also critical during formation of electrocytes. We used immunohistochemical analyses to examine the regeneration of skeletal muscle and electric organ in the presence or absence of innervation. We found that blastema formation is a nerve-dependent process because regeneration was minimal when tail amputation and denervation were performed at the same time. Denervation at the onset of myogenesis resulted in the differentiation of both fast and slow muscle fibers. These were fewer in number, but in a spatial distribution similar to controls. However, in the absence of innervation, fast muscle fibers did not progress beyond the formation of closely apposed clusters, suggesting that innervation is required for their fusion and subsequent transdifferentiation into electrocytes. This study contributes further to our knowledge of the influence of innervation on cell differentiation in the myogenic lineage. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 391–402, 2002

The electric organ discharge (EOD) of gymnotiform electric fish is controlled by a well-characterized neural circuit in the brainstem and spinal cord. NADPH-diaphorase (NADPH-d) activity was previously found in phase-locking and/or rapidly firing neurons in the electromotor and electrosensory systems of Apteronotus leptorhynchus [Turner and Moroz, 1995]. These findings suggested that nitric oxide synthase (NOS) is expressed in these neurons and may regulate their precise, high frequency firing. We extended these results by examining the distribution of both NADPH-d activity and NOS-like immunoreactivity (NOS-lir) in the electromotor systems of four gymnotiform species that differ in the frequency and modulation of their EODs. NOS-lir colocalized with NADPH-d staining throughout the electromotor system, indicating that NADPH-d is a faithful indicator of NOS in this system. The distribution of NOS-lir and NADPH-d was similar in the electromotor systems of all four species in this study, with one exception: NOS and NADPH-d staining was consistently less intense in pacemaker and relay cells in Sternopygus macrurus, which produces low frequency EODs, than in the three other species that produce higher frequency EODs. This species difference in NOS expression in the pacemaker nucleus may be related to species differences either in EOD frequency or in modulations of the EOD (e.g., the jamming avoidance response). In Apteronotus species, NOS-lir and NADPH-d were concentrated in bands along the axons of their nerve-derived electric organs. These bands corresponded to regions surrounded by little or no staining with a Schwann cell-specific antibody, suggesting that the NOS-positive regions lie near nodes of Ranvier. In Sternopygus and Eigenmannia, the innervated, posterior membranes of muscle-derived electrocytes were more intensely labeled for NADPH-d and NOS than inexcitable portions of the membrane. Thus, in both muscle- and nerve-derived electric organs, NOS is concentrated near excitable membranes. These results indicate that NOS is well-positioned within the electromotor system to regulate the frequency, precision, amplitude, and waveform of EODs.

The electric organ has evolved independently from muscle in at least six lineages of fish. How does a differentiated muscle cell change its fate to become an electrocyte? Is the process by which this occurs similar in different lineages? We have begun to answer these questions by studying the formation and maintenance of electrocytes in the genus Sternopygus, a weakly electric teleost. Electrocytes arise from the fusion of fully differentiated muscle fibers, mainly those expressing fast isoforms of myosin. Electrocytes briefly co-express sarcomeric proteins, such as myosin and tropomyosin, and keratin, a protein not found in mature muscle. The sarcomeric proteins are subsequently down-regulated, but keratin expression persists. We investigated whether the maintenance of the electrocyte phenotype depends on innervation. We found that, after spinal cord transection, which silences the electromotor neurons that innervate the electrocytes, or destruction of the spinal cord, which denervates the electrocytes, mature electrocytes re-express sarcomeric myosin and tropomyosin, although keratin expression persists. Ultrastructural examination of denervated electrocytes revealed nascent sarcomeres. Thus, the maintenance of the electrocyte phenotype depends on neural activity.

The electric organ (EO) of the weakly electric fishSternopygus macrurusderives from striated myofibers that fuse and suppress many muscle properties. Mature electrocytes are larger than muscle fibers, do not contain sarcomeres, or express myosin heavy chain (MHC) or tropomyosin. Furthermore, electrocytes express keratin, a protein not expressed in muscle. InS. macrurusthe EO is driven continuously at frequencies higher than those of the intermittently active skeletal muscle. The extent to which differences in EO and muscle phenotype are accounted for by activity patterns, or innervation per se, was determined by assessing the expression of MHC, tropomyosin, and keratin 2 and 5 weeks after the elimination of (1) activity patterns by spinal transection or (2) all synaptic input by denervation.Immunohistochemical analyses showed no changes in muscle fiber phenotypes after either experimental treatment. In contrast, the keratin-positive electrocytes revealed an upregulation of MHC and tropomyosin. Nearly one-third of all electrocytes expressed MHC (35%) and tropomyosin (25%) 2 weeks after spinal transection, whereas approximately two-thirds (61%) expressed MHC 2 weeks after denervation. After 5 weeks of denervation or spinal transection, all electrocytes contained MHC and tropomyosin. Newly formed sarcomere clusters also were observed in denervated electrocytes. The MHC expressed in electrocytes corresponded to that present in a select population of muscle fibers, i.e., type II fibers. Thus, the elimination of electrical activity or all synaptic input resulted in a partial reversal of the electrocyte phenotype to an earlier developmental stage of its myogenic lineage.

To examine the influence of a motoneuron in maintaining the phenotype of the muscle fibers it innervates, myosin heavy chain (MHC) expression, succinate dehydrogenase (SDH) activity, and cross-sectional area (CSA) of a sample of fibers belonging to a motor unit were studied in the cat tibialis anterior 6 mo after the nerve branches innervating the anterior compartment were cut and sutured near the point of entry into the muscle. The mean, range, and coefficient of variation for the SDH activity and the CSA for both motor unit and non-motor unit fibers for each MHC profile and from each control and each self-reinnervated muscle studied was obtained. Eight motor units were isolated from self-reinnervated muscles using standard ventral root filament testing techniques, tested physiologically, and compared with four motor units from control muscles. Motor units from self-reinnervated muscles could be classified into the same physiological types as those found in control tibialis anterior muscles. The muscle fibers belonging to a unit were depleted of glycogen via repetitive stimulation and identified in periodic acid-Schiff-stained frozen sections. Whereas muscle fibers in control units expressed similar MHCs, each motor unit from self-reinnervated muscles contained a mixture of fiber types. In each motor unit, however, there was a predominance of fibers with the same MHC profile. The relative differences in the mean SDH activities found among fibers of different MHC profiles within a unit after self-reinnervation and those found among fibers in control muscles were similar, i.e., fast-2 < fast-1 < or = slow MHC fibers.(ABSTRACT TRUNCATED AT 250 WORDS)