Myocyte Dedifferentiation Drives Extraocular Muscle Regeneration in Adult Zebrafish.

Binocular vision requires intricate control of eye movement by 6 pairs of extraocular muscles (EOMs). Congenital and acquired strabismus secondary to EOM dysfunction cause misaligned binocular inputs, which can lead to amblyopia (neurologic vision loss) or diplopia, collectively affecting as much as 5% to 10% of the US population.1,2 More broadly, skeletal muscle injury and degenerative conditions are common and debilitating and represent important causes of morbidity and mortality worldwide.3,4 Mammalian myonuclei are defined as being postmitotic, and muscle growth and repair are achieved by myogenic progenitor cells termed satellite cells.5–11 These cells are identified by expression of the paired box transcription factor family members Pax7 and/or Pax3. Pax7 is expressed by most adult muscle satellite cells, whereas Pax3 is present only in satellite cells of particular muscles and is transiently expressed by activated satellite cells.12–15 Muscle injury can be a focal injury (e.g., stab wound) or a major injury with significant tissue loss and every degree of injury in between. Response to focal injury would require a local repair process, whereas significant tissue loss would require more extensive tissue generation (i.e., “whole-organ” regeneration). In mammals, muscle repair following focal injury involves the proliferation of satellite cells to form myoblasts that express the transcription factor MyoD. Some myoblasts maintain MyoD expression, downregulate Pax7, and commit to differentiation by activation of myogenin. These differentiated myoblasts fuse to form myocytes. Other myoblasts maintain Pax7 expression, downregulate MyoD, and eventually withdraw from the cell cycle to maintain the satellite cell population of the muscle.14,16,17 In contrast, whole-organ muscle regeneration is much more limited in mammals despite the presence of satellite cells. In salamanders, limb amputation is followed by limb regeneration in which Pax7-positive cells play an important role in some but not all salamander species.18 Pax7-positive satellite cells have also been described in mammalian EOMs.19 Additionally, progenitor cells that express PITX2 have been described.20 EOMs are a specialized form of skeletal muscle derived from branchial and cephalic mesoderm,21–23 and EOM satellite cells may have certain distinguishing features compared to their somitic counterparts. However, published results are somewhat inconsistent. Some results suggest that the EOM stem cell niche is protected from aging and disease24,25 and maintains proliferative potential longer than somitic muscles.26 Other results argue that aged EOM stem cells lose their ability to fuse to form multinucleated myotubes.26 This disagreement in published conclusions may be the result of using different species and/or different experimental approaches. To investigate the potential for adult whole-organ skeletal muscle regeneration, we exploited the well-documented regenerative capacity of adult zebrafish. With its genome nearly fully sequenced and with well-developed molecular tools, the zebrafish (Danio rerio) has become an important model for human disease research. In particular, adult zebrafish have robust regenerative capacity in organs as diverse as the brain, retina, fins, and heart.27–32 In regenerating complex tissues, zebrafish use both resident stem cells (e.g., melanocyte precursors in fins33) and lineage-restricted dedifferentiated cells (e.g., cardiac myocytes,34 retinal Muller glia,31,35 and osteoblasts32,36), suggesting that zebrafish might be an ideal model for studying de novo whole-organ muscle regeneration. Our model uses a large EOM myectomy in sexually mature adult zebrafish. We focused on the lateral rectus (LR) muscle because of its anatomic isolation from the other EOMs37 and its surgical accessibility and the ability to assess its function using horizontal gaze eye movement. Here we report that, following myectomy of approximately 50% of the LR muscle, adult zebrafish regenerated an anatomically correct and functional muscle. Unlike embryonic EOM development, the regeneration process appears to be independent of pax7-positive satellite cells, as classic satellite cells are not detectable in zebrafish adult EOMs by electron microscopy or immunofluorescence techniques. Instead, our data reveal that residual EOM myocytes undergo dedifferentiation to form myoblasts, followed by robust myoblast proliferation and redifferentiation into functional muscle fibers. We propose that the ability of residual EOM myocytes to dedifferentiate following severe injury underlies their whole-organ regenerative capacity in zebrafish, compared with other species in which dedifferentiation is actively blocked. Furthermore, adult zebrafish EOM regeneration via dedifferentiation offers a powerful model for studying the mechanistic underpinnings of cellular reprogramming and dedifferentiation.