Fundamental Differences in Dedifferentiation and Stem Cell Recruitment during Skeletal Muscle Regeneration in Two Salamander Species

Abstract

Salamanders regenerate appendages via a progenitor pool called the blastema. The cellular mechanisms underlying regeneration of muscle have been much debated but have remained unclear. Here we applied Cre-loxP genetic fate mapping to skeletal muscle during limb regeneration in two salamander species, Notophthalmus viridescens (newt) and Ambystoma mexicanum (axolotl). Remarkably, we found that myofiber dedifferentiation is an integral part of limb regeneration in the newt, but not in axolotl. In the newt, myofiber fragmentation results in proliferating, PAX7(-) mononuclear cells in the blastema that give rise to the skeletal muscle in the new limb. In contrast, myofibers in axolotl do not generate proliferating cells, and do not contribute to newly regenerated muscle; instead, resident PAX7(+) cells provide the regeneration activity. Our results therefore show significant diversity in limb muscle regeneration mechanisms among salamanders and suggest that multiple strategies may be feasible for inducing regeneration in other species, including mammals.

Neuregulin-1 signaling is essential for nerve-dependent axolotl limb regeneration

“…Although NRG1 is the first protein to our knowledge that has been shown to be capable of rescuing regeneration to digits in the axolotl limb, these findings do not rule out the possibility of other factors playing a crucial role in this process. Newt anterior gradient protein has been shown to rescue regeneration in denervated newt limbs (Kumar et al, 2007), and despite some prominent species differences between axolotls and newts, which demonstrate a different recovery response to denervation (Liversage and McLaughlin, 1983) as well as a phylogenetically unique method of regenerating muscular tissues (Sandoval-Guzman et al, 2014;Tanaka et al, 2016), further exploration of the relationship between these two signaling pathways is necessary in order to characterize fully the underlying cause of nerve dependency in the axolotl limb. Given the conserved role of NRG1/ErbB2 signaling in the peripheral nerves as well as the burgeoning evidence of its necessity in other animal models of cardiac (Bersell et al, 2009;D'Uva et al, 2015;Gemberling et al, 2015) and peripheral nerve (Fricker et al, 2011;Ronchi et al, 2013Ronchi et al, , 2015 regeneration, elucidating the function and mechanism of this signaling pathway in the axolotl may have far-reaching impacts on the field of regenerative medicine.…”

Mechanisms of urodele limb regeneration

“…Histolysis liberates fibroblasts from the dermis, interstitial connective tissue of muscle, periosteum, and nerve sheath, as well as Schwann cells from the peripheral nerves. Myofibers fragment at their cut ends and break up into mononucleate cells while simultaneously releasing Pax7 + muscle stem cells, the satellite cells (Hay, 1959; Sandoval‐Guzman et al., 2014; Thornton, 1938a, b). MMPs also prevent reassembly of a basement membrane, thereby ensuring contact between the wound epidermis and the underlying tissues.…”

“…Satellite cells are the source of regenerated muscle in larval and metamorphosed axolotls (Sandoval‐Guzman et al., 2014). The larval newt limb also mobilizes satellite cells to regenerate muscle, but the adult newt limb switches to dedifferentiation of mononucleate myofiber fragments as the primary source of muscle progenitors (Tanaka et al., 2016; Young, Bailey, Markwald, & Dalley, 1985).…”

“…The same structure/function/regeneration correlation extends to the urodele and zebrafish heart versus the mammalian heart. Furthermore, comparative studies suggest that differences in regenerative capacity and mechanism among species or different developmental stages are influenced by fundamental traits such as body size, aging, and growth pattern (Seifert et al, 2012), making it important to consider experimental results with reference to these traits, as has been shown for muscle contribution to the blastema (Sandoval‐Guzman et al., 2014). …”

Limb regeneration in a direct‐developing terrestrial salamander, Bolitoglossa ramosi (Caudata: Plethodontidae)

“…Recently, it has been shown that these species have different mechanisms to rebuild tissues during limb regeneration; for example, muscle regeneration in axolotls is led by satellite cells, whereas newt muscles go through a process of dedifferentiation to generate proliferating cells (Sandoval‐Guzmán et al., 2014). The capacity for lens regeneration also changes: whereas axolotls can regenerate a lens only during early developmental stages, newts sustain this ability throughout adulthood (Eguchi et al., 2011).…”