The ability to form a regeneration blastema, which leads to the epimorphic regeneration of complex body structures, is restricted to some amphibians and fish among vertebrates (Poss et al., 2003
). A conundrum of regenerative biology is why mammals, with a few exceptions, do not form a blastema or a blastema-like structure despite the fact that they can functionally repair some tissues, such as skeletal muscle (Charge and Rudnicki, 2004
) and liver (Fausto and Campbell, 2003
). Of particular interest is whether the generation of progenitor cells during epimorphic regeneration in salamander and during mammalian tissue repair proceeds by the activation of different or overlapping mechanisms. A unique feature of blastema formation in salamanders is the process of dedifferentiation of stump tissues that follows appendage removal. The possibility to induce blastema formation and regeneration in mammals through the activation of a comparable dedifferentiation program has been proposed (Hughes, 2001
; Bryant et al., 2002
; Stocum, 2004
). This is especially valid for skeletal muscle tissue because dedifferentiating skeletal muscle is a significant source of blastema progenitors. Although the potential role of stem cells in blastema formation has been suggested (Corcoran and Ferretti, 1999
; Carlson, 2003
; Odelberg, 2004
), no such cells have been previously identified in the newt limb. Hence, it is still not clear whether the term dedifferentiation solely refers to the reversal of the differentiated state of mature cells, to the activation of stem cells in the disorganizing tissues, or to a combination of these two definitions. If both processes coexist, the quantitative aspects of their relative contribution in vivo remain to be elucidated.
Our data clearly show that satellite cells, which are comparable to mammalian skeletal muscle stem cells, exist in newt skeletal muscle as well. First, we found that newt satellite cells or their progeny express molecular markers, such as Pax7, M-cadherin, and MyoD, all of which are expressed by mammalian satellite cells or their progeny as well (Zammit and Beauchamp, 2001
). Second, when we isolated single myofibers a satellite cell population was copurified, despite the presence of an additional basal lamina between the satellite cell and sarcolemma. Third, similar to the mammalian myofiber cultures, we observed that satellite cell activation occurred that was characterized by cell cycle reentry and proliferation of the satellite cell progeny population. Finally, we showed that the satellite cell progeny population in newts is multipotent, which has also been observed in mammals (Asakura et al., 2001
; Wada et al., 2002
; Shefer et al., 2004
Thus, the results indicate that newts do not represent an exception in the vertebrate phyla, and like other amphibians (Mauro, 1961
; Gargioli and Slack, 2004
) and mammals they also contain Pax7+
stem cells in their skeletal muscle tissue. However, the additional basement membrane that separates newt satellite cells from the sarcolemma may reflect that newt satellite cells are in some respect evolutionary intermediates between interstitial stem cells and satellite cells, which were found to be separate populations in mammals (Asakura et al., 2002
; Tamaki et al., 2002
). Identification of further stem cell populations in newt skeletal muscle, along with functional studies, could address this issue.
The satellite cell progeny population was able to adopt nonmyogenic fates in vitro and they incorporated into the regeneration blastema after intramuscular injection before amputation. We also noted a contribution to the epidermis and detected satellite cell progeny within newly formed cartilage tissue. The observed multipotentiality of satellite cell progeny does not directly address the question of whether activated satellite cells adopt divergent fates without in vitro expansion. However, the onset of tissue-specific molecular differentiation programs and the large number of satellite cell progeny within various tissues, which did not alter the speed and mode of regeneration, suggest that the integrated satellite cell progeny are functional. Furthermore, lineage shifting across germ layer boundaries has been shown to occur during salamander tail regeneration (Echeverri and Tanaka, 2002
). Clearly, additional experiments are required to assess the plasticity of satellite cells in vivo and to establish whether metaplasia characterizes salamander limb regeneration. Nevertheless, in light of the available observations, a plausible hypothesis is that skeletal muscle dedifferentiation results in a significant contribution by satellite cells to the blastema and to the regenerate. Pax7+
cells are also found in the blastema of the regenerating axolotl tail (Schnapp et al., 2005
) and tail regeneration in the Xenopus laevis
tadpole also involves satellite cell activation (Gargioli and Slack, 2004
). These observations further suggest an important role of satellite cells in the regeneration of missing body parts in vertebrates.
In a study similar to our own, Kumar et al. (2004)
showed that limb myofibers isolated from axolotl larvae undergo cellularization and fragmentation. The authors noted that only 3.5% of the myofibers contained the satellite type of cells and that these were not observed in their skeletal muscle fiber plasticity model. We saw that 86% of the isolated myofibers contained satellite cells and that only satellite cell progeny proliferated in our culture system, although we could not detect any sign of proliferating progeny that could have been derived by cellularization of the myofiber. At present, it is unclear whether the discrepancies between our observations and the model presented by Kumar et al. (2004)
reflect phylogenetic or ontogenetic differences, or are caused by dissimilarities in the experimental paradigms. However, both studies underpin the necessity to further assess the quantitative aspects and functional relevance of satellite cell activation that leads to multipotent progeny on one hand and cellularization and/or fragmentation of the syncytium on the other during limb regeneration.
Our results show that epimorphic limb regeneration activates such programs, which lead to regeneration of muscle tissue in mammals after injury. Mammalian skeletal muscle responds to various challenges, such as stretching or mechanical damage, by activating a proliferation program in satellite cells that is followed by differentiation and fusion into myotubes and into myofibers. In this context, it is interesting to note the study by Echeverri et al. (2001)
, which showed that amputation as such was not sufficient to produce blastema progenitors. Instead, a mechanical stimulus (minor clipping of the muscle fiber) was required for the generation of progeny from dedifferentiating axolotl tail muscle in vivo (Echeverri et al., 2001
). The exact identity of signals that link tissue injury to blastema formation needs to be elucidated, as it may reveal key aspects of blastema formation involving both myofiber fragmentation and concomitant stem cell activation. Formation of a blastema-like structure, although a rare event, is possible in mammals, as exemplified by the healing capacity of MRL mice and by the seasonal regeneration of deer antlers (Gourevitch et al., 2003
; Price et al., 2005
). The question is how blastema formation is induced in mammals and how it can be promoted. We propose skeletal muscle satellite cells as a potential target in the promotion of mammalian blastema formation.