The latent dedifferentiation capacity of newt limb muscles is unleashed by a combination of metamorphosis and body growth

Abstract

Newts can regenerate their limbs throughout their life-span. Focusing on muscle, certain species of newts such as Cynops pyrrhogaster dedifferentiate muscle fibers in the limb stump and mobilize them for muscle creation in the regenerating limb, as they grow beyond metamorphosis. However, which developmental process is essential for muscle dedifferentiation, metamorphosis or body growth, is unknown. To address this issue, we tracked muscle fibers during limb regeneration under conditions in which metamorphosis and body growth were experimentally shifted along the axis of development. Our results indicate that a combination of metamorphosis and body growth is necessary for muscle dedifferentiation. On the other hand, ex vivo tracking of larval muscle fibers revealed that newt muscle fibers have the ability to dedifferentiate independently of metamorphosis and body growth. These results suggest that newt muscle fibers have an intrinsic ability to dedifferentiate, but that metamorphosis and body growth are necessary for them to exhibit this hidden ability. Presumably, changes in the extracellular environment (niche) during developmental processes allow muscle fibers to contribute to limb regeneration through dedifferentiation. This study can stimulate research on niches as well as gene regulation for dedifferentiation, contributing to a further understanding of regeneration and future medical applications.

Newtic1 Is a Component of Globular Structures That Accumulate along the Marginal Band of Erythrocytes in the Limb Blastema of Adult Newt, Cynops pyrrhogaster

“…A strain of C. pyrrhogaster , Toride-Imori, was also used [ 16 ]. The animals were reared at 18–22 °C under natural light conditions until experiments as described previously [ 12 , 13 , 14 , 15 ].…”

“…Animals were anesthetized in 0.1% FA100 (4-allyl-2-methoxyphenol; LF28C054; DS Pharma Animal Health, Osaka, Japan) dissolved in water at room temperature (RT: 22 °C) for 45 min, as described previously [ 12 , 13 , 14 , 15 ].…”

“…To varying degrees, four-limbed vertebrates (tetrapods), including humans, generally have the ability to regenerate lost complex tissues or body parts after trauma early in development, but as they grow and become adults, this ability is reduced or lost, and the deficient areas heal instead by being covered with fibrotic tissue [ 1 , 2 ]. Contrary to this general rule, newts, which belong to a group of the family Salamandridae in urodele amphibians, have the ability to repeatedly regenerate lost body parts, regardless of their age, even after reaching adulthood beyond metamorphosis [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. It is believed that this outstanding regenerative ability of adult newts is based on a mechanism of cellular reprogramming/dedifferentiation that is unique to newts: in adult newts, terminally differentiated somatic cells, which have already lost premature traits such as multipotency factor expression and proliferative activity and have become highly specialized for specific physiological functions, are reprogrammed/dedifferentiate into stem/progenitor-like cells upon trauma.…”

“…It is believed that this outstanding regenerative ability of adult newts is based on a mechanism of cellular reprogramming/dedifferentiation that is unique to newts: in adult newts, terminally differentiated somatic cells, which have already lost premature traits such as multipotency factor expression and proliferative activity and have become highly specialized for specific physiological functions, are reprogrammed/dedifferentiate into stem/progenitor-like cells upon trauma. For example, retinal pigment epithelium (RPE) cells in the eyes are reprogrammed into RPE stem cells for retinal regeneration [ 1 , 6 , 7 , 9 ] while striated muscle fibers (tubular, multinucleated cells with sarcomeres) in the limbs dedifferentiate into mononucleated myogenic cells for muscle regeneration [ 10 , 14 ]. This ability could be interpreted as allowing adult newts to mobilize stem/progenitor-like cells from terminally differentiated somatic cells as regenerative material to complement resident somatic stem/progenitor cells that have reduced their contribution to regeneration as adults [ 14 ].…”

“…For example, retinal pigment epithelium (RPE) cells in the eyes are reprogrammed into RPE stem cells for retinal regeneration [ 1 , 6 , 7 , 9 ] while striated muscle fibers (tubular, multinucleated cells with sarcomeres) in the limbs dedifferentiate into mononucleated myogenic cells for muscle regeneration [ 10 , 14 ]. This ability could be interpreted as allowing adult newts to mobilize stem/progenitor-like cells from terminally differentiated somatic cells as regenerative material to complement resident somatic stem/progenitor cells that have reduced their contribution to regeneration as adults [ 14 ]. Therefore, body part regeneration in adult newts is a useful model system for basic research toward new regenerative medicine combining stem cells and dedifferentiation.…”