Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish

Abstract

Satellite cells, also known as muscle stem cells, are responsible for skeletal muscle growth and repair in mammals. Pax7 and Pax3 transcription factors are established satellite cell markers required for muscle development and regeneration, and there is great interest in identifying additional factors that regulate satellite cell proliferation, differentiation, and/or skeletal muscle regeneration. Due to the powerful regenerative capacity of many zebrafish tissues, even in adults, we are exploring the regenerative potential of adult zebrafish skeletal muscle. Here, we show that adult zebrafish skeletal muscle contains cells similar to mammalian satellite cells. Adult zebrafish satellite-like cells have dense heterochromatin, express Pax7 and Pax3, proliferate in response to injury, and show peak myogenic responses 4–5 days post-injury (dpi). Furthermore, using a pax7a-driven GFP reporter, we present evidence implicating satellite-like cells as a possible source of new muscle. In lieu of central nucleation, which distinguishes regenerating myofibers in mammals, we describe several characteristics that robustly identify newly-forming myofibers from surrounding fibers in injured adult zebrafish muscle. These characteristics include partially overlapping expression in satellite cells and regenerating myofibers of two RNA-binding proteins Rbfox2 and Rbfoxl1, known to regulate embryonic muscle development and function. Finally, by analyzing pax7a; pax7b double mutant zebrafish, we show that Pax7 is required for adult skeletal muscle repair, as it is in the mouse.

Pnrc2 regulates 3’UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation

“…Primers used to assess efficacy of pnrc2 sbMO injection (Fig S1K–L) are listed in Table S4. upf1, rbfox1l , and rbfox2 sbMO doses were performed according to published methods using doses that gave reproducible phenotypic defects matching published results (Wittkopp et al, 2009; Gallagher et al, 2011; Berberoglu et al, 2017). Embryos were incubated at 28.5°C until 6 hours post fertilization (hpf) and then transferred to 25°C thereafter, except for a subset of rbfox1l/rbfox2 double-injected and uninjected control embryos that were incubated at 28.5°C until 24 hpf and subsequently scored for ability to move.…”

“…Interestingly, single upf1 knockdown at published (0.65 ng) or higher doses (2 ng) does not alter her1 mRNA expression, but does induce the expected published phenotypes including neural necrosis and abnormal segment formation (data not shown) (Wittkopp et al, 2009; Anastasaki et al, 2011). As a control, we injected a sub-optimal dose of pnrc2 sbMO (2 ng) with an unrelated MO, rbfox1l sbMO, at the published 6 ng dose (Gallagher et al, 2011; Berberoglu et al, 2017), and observed that her1 mRNA expression is normal (Fig S3), suggesting that her1 misexpression in embryos co-injected with sub-optimal doses of pnrc2 and upf1 sbMOs is a specific effect. Taken together, these results reveal that depletion of Upf1 sensitizes embryos to partial loss of Pnrc2.…”

“…The upf1 sbMO, 5′-TTTTGGGAGTTTATACCTGGTTGTC-3′, was published previously (Wittkopp et al, 2009). The rbfox1l sbMO, 5′-GCATTTGTTTTACCCCAAACATCTG-3′, and rbfox2 sbMO, 5′-TATAATGCTTTATATACCCCGAACA-3′, was published previously (Gallagher et al, 2011; Berberoglu et al, 2017). Morpholinos were diluted to 0.1–2 ng/nl in 0.2M KCl and 0.1% phenol red and injected into the yolk of 1-cell stage embryos.…”

RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes

“…In zebrafish, the PAX7 orthologue is duplicated in two copies; pax7a and pax7b. pax7a expressing cells act to initiate myofiber formation post-injury, whereas pax7a/b expressing cells are required for myofiber growth [30]. Significant down regulation of both pax7a and b was observed during the larval stage in ddx27 mutant zebrafish.…”

Single cell dynamics of embryonic muscle progenitor cells in zebrafish

“…cmet + cells are capable of selfrenewal and contribute to muscle regeneration, suggesting that they are functionally equivalent to mammalian satellite cells (Gurevich et al, 2016). Recent work has also shown that Pax3/7-expressing satellite-like cells in adult zebrafish are essential for the regeneration after muscle injury (Berberoglu et al, 2017). Therefore, pax7 labels multiple MPC populations at different developmental stages in zebrafish: (1) embryonic MPCs in the dermomyotome on the surface of the somite; (2) fiber-associated deep myotomal cells (including cmet + cells) in larval zebrafish; and (3) satellite-like cells in adult zebrafish.…”