Fgf regulates dedifferentiation during skeletal muscle regeneration in adult zebrafish

Abstract

Fibroblast growth factors (Fgfs) regulate critical biological processes such as embryonic development, tissue homeostais, wound healing, and tissue regeneration. In zebrafish, Fgf signaling plays an important role in the regeneration of the spinal cord, liver, heart, fin, and photoreceptors, although its exact mechanism of action is not fully understood. Utilizing an adult zebrafish extraocular muscle (EOM) regeneration model, we demonstrate that blocking Fgf receptor function using either a chemical inhibitor (SU5402) or a dominant-negative transgenic construct (dnFGFR1a:EGFP) impairs muscle regeneration. Adult zebrafish EOMs regenerate through a myocyte dedifferentiation process, which involves a muscle-to-mesenchyme transition and cell cycle reentry by differentiated myocytes. Blocking Fgf signaling reduced cell proliferation and active caspase 3 levels in the regenerating muscle with no detectable levels of apoptosis, supporting the hypothesis that Fgf signaling is involved in the early steps of dedifferentiation. Fgf signaling in regenerating myocytes involves the Mapk/Erk pathway: inhibition of Mek activity with U0126 mimicked the phenotype of the Fgf receptor inhibition on both muscle regeneration and cell proliferation, and activated ERK (p-ERK) was detected in injured muscles by immunofluorescence and western blot. Interestingly, following injury, ERK2 expression is specifically induced and activated by phosphorylation, suggesting a key role in muscle regeneration. We conclude that the critical early steps of myocyte dedifferentiation in EOM regeneration are dependent on Fgf signaling.

Neuromuscular Activity Induces Paracrine Signaling and Triggers Axonal Regrowth after Injury in Microfluidic Lab-On-Chip Devices

“…The latter has recently been described as being secreted by muscular cells to induce vascularization after injury [45] and was also described as boosting regeneration in dorsal root ganglia (DRGs) [58], Fgfb [57] implicated in the development and regeneration of spinal cord, liver, heart, and photoreceptors from the zebrafish [59]. Moreover, released by the muscle in an autocrine fashion after injury [60] is the glial-derived neurotrophic factor GDNF, whose direct muscle delivery with human mesenchymal stem cells improves motor neuron survival and function in an amyotrophic lateral sclerosis (ALS) model [61]. In addition, motor neuron survival can be promoted by the muscle-specific overexpression of GDNF or exogenous GDNF [62][63][64][65][66][67], which is why in embryonic motor neuron culture GDNF is added as a survival factor.…”

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

“…Although pax7 -driven GFP protein perdurance supports the idea that satellite-like cells contribute to new muscle after injury, it is not a definitive demonstration, and it is possible that new muscle forms by satellite cell-independent mechanisms. Some adult zebrafish tissues, like heart, fin, extraocular muscle, and retina, regenerate via dedifferentiation of mature cell types without using a specific precursor population (Geurtzen et al, 2014; Jopling et al, 2010; Knopf et al, 2011; Poss et al, 2002; Ramachandran et al, 2010; Saera-Vila et al, 2015; Saera-Vila et al, 2016; Sousa et al, 2011; Stewart et al, 2012; Wan et al, 2012). Particularly relevant to our study, post-injury regeneration of adult extraocular skeletal muscle uses dedifferentiation (Saera-Vila et al, 2015; Saera-Vila et al, 2016), demonstrating that some skeletal muscle types are repaired using satellite cell-independent mechanisms.…”

“…Zebrafish are competent in both regeneration (e.g., regeneration of the heart; Poss et al, 2002) and repair (e.g., healing after focal needle-stick injury; Gurevich et al, 2016; Knappe et al, 2015; März et al, 2011; Rowlerson et al, 1997; Seger et al, 2011). A few studies have examined skeletal muscle repair (Rowlerson et al, 1997; Tee et al, 2012) and regeneration (Saera-Vila et al, 2015; Saera-Vila et al, 2016) after injury in adult zebrafish. The work from Rowlerson and colleagues (1997) was performed before the identification of satellite cell markers such as Pax7 and Pax3, and thus did not address whether adult zebrafish possess myogenic progenitor cells that function as satellite cells.…”

“…A second study (Tee et al, 2012) observed Pax7-positive cells after needle-stick injury of adult zebrafish trunk musculature; however, only one time-point was examined and the contribution of Pax7-expressing cells to muscle repair was not assessed. Most recently, regeneration of extraocular muscle was found to occur through Fgf-regulated myocyte dedifferentiation that does not employ classic Pax7-positive satellite-like cells (Saera-Vila et al, 2015; Saera-Vila et al, 2016). Here, we have undertaken a comprehensive study to define the cells employed and steps involved during trunk skeletal muscle repair.…”

Fibroblast growth factors: key players in regeneration and tissue repair

“…Similar to their roles in heart repair, FGFs are also key players in skeletal muscle regeneration in adult zebrafish. In an extraocular muscle injury model, in which regeneration occurs via a myocyte de-differentiation process and re-entry of the de-differentiated cells into the cell cycle, the blocking of FGFR signaling with a receptor kinase inhibitor or with a dominant-negative FGFR strongly impairs muscle regeneration (Saera-Vila et al, 2016).…”