Macrophages provide a transient muscle stem cell niche via NAMPT secretion.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

Stem cells in skeletal muscle growth and regeneration in amniotes and teleosts: Emerging themes.

Skeletal muscle is a contractile, postmitotic tissue that retains the capacity to grow and regenerate throughout life in amniotes and teleost. Both muscle growth and regeneration are regulated by obligate tissue resident muscle stem cells. Given that considerable knowledge exists on the myogenic process, recent studies have focused on examining the molecular markers of muscle stem cells, and on the intrinsic and extrinsic signals regulating their function. From this, two themes emerge: firstly, muscle stem cells display remarkable heterogeneity not only with regards to their gene expression profile, but also with respect to their behavior and function; and secondly, the stem cell niche is a critical regulator of muscle stem cell function during growth and regeneration. Here, we will address the current understanding of these emerging themes with emphasis on the distinct processes used by amniotes and teleost, and discuss the challenges and opportunities in the muscle growth and regeneration fields. This article is characterized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Early Embryonic Development > Development to the Basic Body Plan Vertebrate Organogenesis > Musculoskeletal and Vascular.

Muscle precursor cell movements in zebrafish are dynamic and require Six family genes.

Muscle precursors need to be correctly positioned during embryonic development for proper body movement. In zebrafish, a subset of hypaxial muscle precursors from the anterior somites undergo long-range migration, moving away from the trunk in three streams to form muscles in distal locations such as the fin. We mapped long-distance muscle precursor migrations with unprecedented resolution using live imaging. We identified conserved genes necessary for normal precursor motility (six1a, six1b, six4a, six4b and met). These genes are required for movement away from somites and later to partition two muscles within the fin bud. During normal development, the middle muscle precursor stream initially populates the fin bud, then the remainder of this stream contributes to the posterior hypaxial muscle. When we block fin bud development by impairing retinoic acid synthesis or Fgfr function, the entire stream contributes to the posterior hypaxial muscle indicating that muscle precursors are not committed to the fin during migration. Our findings demonstrate a conserved muscle precursor motility pathway, identify dynamic cell movements that generate posterior hypaxial and fin muscles, and demonstrate flexibility in muscle precursor fates.

Fluorescence-Activated Cell Sorting of Larval Zebrafish Muscle Stem/Progenitor Cells Following Skeletal Muscle Injury.

This chapter describes a protocol for the isolation of larval zebrafish muscle stem/progenitor cells by fluorescence-activated cell sorting (FACS). This method has been successfully applied to isolate pax3a expressing cells 3 days following needle stab skeletal muscle injury. The cell sorting strategy described here can easily be adapted to any cell type at embryonic or larval stages. RNA extracted from the sorted cells can be used for subsequent downstream applications such as quantitative PCR (qPCR), microarrays, or next generation sequencing.

Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences.

OBJECTIVE: Repeat expansion of polyglutamine tracks leads to a group of inherited human neurodegenerative disorders. Studying such repetitive sequences is required to gain insight into the pathophysiology of these diseases. PCR-based manipulation of repetitive sequences, however, is challenging due to the absence of unique primer binding sites or the generation of non-specific products. RESULTS: We have utilised the degeneracy of the genetic code to generate a polyglutamine sequence with low repeat similarity. This strategy allowed us to use conventional PCR to generate multiple constructs with approximately defined numbers of glutamine repeats. We then used these constructs to measure the in vivo variation in autophagic degradation activity related to the different numbers of glutamine repeats, providing an example of their applicability to study repeat expansion diseases. Our simple and easily generalised method of generating low repetition DNA sequences coding for uniform stretches of amino acid residues provides a strategy for generating particular lengths of polyglutamine tracts using standard PCR and cloning protocols.

Stem cell dynamics in muscle regeneration: Insights from live imaging in different animal models.

In recent years, live imaging has been adopted to study stem cells in their native environment at cellular resolution. In the skeletal muscle field, this has led to visualising the initial events of muscle repair in mouse, and the entire regenerative response in zebrafish. Here, we review recent discoveries in this field obtained from live imaging studies. Tracking of tissue resident stem cells, the satellite cells, following injury has captured the morphogenetic dynamics of stem/progenitor cells as they facilitate repair. Asymmetric satellite cell division generated a clonogenic progenitor pool, providing in vivo validation for this mechanism. Furthermore, there is an emerging role of stem/progenitor cell guidance at the injury site by cellular protrusions. This review concludes that live imaging is a critical tool for discovering the distinct processes that occur during regeneration, emphasising the importance of imaging in diverse animal models to capture the entire scope of stem cell functions. Also see the Video Abstract. Link to: https://youtube/tgUHSBD1N0g.

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

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.