Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo

Dividing asymmetrically to fix muscle Resident tissue stem cells called satellite cells repair muscle after injury. However, how satellite cells operate inside living tissue is unclear. Gurevich et al. exploited the optical clarity of zebrafish larvae and used a series of genetic approaches to study muscle injury. After injury, satellite cells divide asymmetrically to generate a progenitor pool for muscle replacement and at the same time “self-renew” the satellite stem cell. This results in regeneration that is highly clonal in nature, validating many decades of in vitro analyses examining the regenerative capacity of skeletal muscle. Science, this issue p. 136 The visualization of myogenic repair in zebrafish muscle reveals a dynamic regeneration process in living animals. INTRODUCTION Mammalian skeletal muscle harbors tissue-specific stem cells that are triggered to replace damaged fibers after injury. Genetic ablation of satellite cells in the mouse results in a failure to regenerate muscle, which indicates that these cells are the major (and possibly only) mediators for repair of skeletal muscle. Further evidence for the central role of satellite cells in muscle regeneration comes from transplantation experiments with genetically marked cells, which demonstrate that satellite cells are highly proliferative myogenic precursors capable of self‐renewal and the resumption of quiescence, properties deemed important in a cell population responsible for muscle repair. Considerable in vitro evidence, derived from cultured fibers and myoblasts, is suggestive of a role for asymmetric division in generating both a self-renewing “immortal” stem cell and a differentiation-competent progenitor cell that proliferates and ultimately replaces damaged muscle. However, asymmetric division of satellite cells has not been documented in vivo. Furthermore, considerable doubt remains over how accurately in vitro studies can model satellite cell behavior, given that the isolation and culture of individual muscle fibers and cells stimulates satellite cell proliferation. Finally, it is not clear whether the environment an activated satellite cell encounters in a single fiber explant, or in culture, mimics the molecular and biophysical architecture of a regenerating muscle injury in vivo. Consequently, what role, if any, the wound environment itself plays in regeneration and self-renewal is difficult to address in these systems. RATIONALE Using the optical clarity and genetic tractability of the zebrafish system, we developed tools to track and image the regeneration of living muscle tissue after injury. Marking muscle stem and progenitor cells with transgenes and using long-term imaging and lineage-tracing modalities enabled us to visualize cell movements and behaviors during regeneration in vivo. RESULTS In vivo cell tracking permitted high-resolution imaging of the entire process of muscle regeneration, from injury to fiber replacement. Using this approach, we were able to determine the morphological, cellular, and genetic basis for zebrafish muscle regeneration. Our analysis identified a stem cell niche in the zebrafish myotome that is equivalent to the mammalian satellite cell system, revealing that this evolutionarily ancient stem cell is probably present throughout the vertebrate phylogeny. Complex interactions were observed between satellite cells and both injured and uninjured fibers within the wound environment. Among the most notable of these was the identification of filopodia-like projections, emanating from uninjured fibers, which adhere to and “lasso” the activated satellite cell to guide it to the wound edge. Furthermore, we documented the in vivo occurrence of asymmetric satellite cell division, a process that drives both self-renewal and regeneration via a clonally restricted progenitor pool. CONCLUSION Asymmetric divisions occur during in vivo muscle regeneration to generate clonally related progenitors required for muscle repair. This finding resolves a long-term debate surrounding the existence of this mechanism of stem cell self-renewal and muscle repair in vivo. Our results also reveal the highly dynamic nature of the wound environment, where uninjured fibers at the wound edge play a crucial role in directing differentiating progenitors to regions of the wound that are most in need of new fiber addition. Mechanism of in vivo muscle repair. (A to C) Muscle regeneration is clonal. Regenerating fibers (outlined in white) express the same color after fluorescent lineage tracing, indicating clonal derivation from a single stem cell. Sagittal, transverse, and coronal sections are shown in (A) to (C), respectively. (D) Regeneration dynamics in vivo. Quiescent satellite cells, activated upon injury, undergo asymmetric division, which results in self-renewing or proliferating cells. Proliferative cells undergo myogenesis to generate de novo immature fibers. Skeletal muscle is an example of a tissue that deploys a self-renewing stem cell, the satellite cell, to effect regeneration. Recent in vitro studies have highlighted a role for asymmetric divisions in renewing rare “immortal” stem cells and generating a clonal population of differentiation-competent myoblasts. However, this model currently lacks in vivo validation. We define a zebrafish muscle stem cell population analogous to the mammalian satellite cell and image the entire process of muscle regeneration from injury to fiber replacement in vivo. This analysis reveals complex interactions between satellite cells and both injured and uninjured fibers and provides in vivo evidence for the asymmetric division of satellite cells driving both self-renewal and regeneration via a clonally restricted progenitor pool.