Journal of Molecular Cell Biology Advance Access published online on September 30, 2009
Journal of Molecular Cell Biology, doi:10.1093/jmcb/mjp027
Embryonic vs. Adult Myogenesis: Challenging the ‘Regeneration Recapitulates Development’ Paradigm
Department of Bioengineering, University of California, Berkeley, 174 Stanley Hall, Berkeley, CA 94720-1762, USA
* Correspondence to: Irina Conboy, E-mail: iconboy{at}berkeley.edu
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A popular theory in the stem cell field is that ‘regeneration recapitulates development’, or that adult stem cells function similarly to embryonic ones. In a recent Nature article, Lepper et al. questioned this approach, highlighting the differences in requirements for Pax7 during myogenesis for embryonic, juvenile and adult muscle.
A popular theory in the stem cell field is that regeneration recapitulates development. The logic behind this theory is that adult stem cells derive from cells that remain in an embryonic-like state instead of being fully differentiated during development. These stem cells then differentiate in a manner similar to their embryonic brethren to repair damaged tissues. In accordance with this theory, the functions of many genes and proteins involved in stem cell regulation have been identified and analyzed using developmental studies (Silkstone et al., 2008; Brochhausen et al., 2009). Although this approach can yield valuable insight into the roles of specific genes in stem cell function, it has its limitations. Adult tissues and organs do not necessarily display the same gene expression profiles as their embryonic, fetal or juvenile counterparts. As such, the same proteins in adult tissues and pre-adult tissues may have different partners with which to interact or differential access to areas of chromatin which may have been opened or closed through epigenetic modifications that occur during development (Mohn and Schubeler, 2009). Furthermore, adult tissues, which are mostly post-mitotic and have quiescent organ stem cells, certainly differ from developing embryonic organs in both their etiology and properties. Although both exhibit regenerative capacity, only in the latter do active cell proliferation and differentiation co-exist. In this regard, neonatal or juvenile organs represent an intermediate stage where some of the fetal-derived organ-formation and growth continues, whereas the adult program of post-mitotic tissue maintenance is being set forth. Hints of these differences first emerged with a report hallmarking different needs for β-catenin signaling during embryonic and fetal myogenesis (Hutcheson et al., 2009).
In their recently published Nature article (Lepper et al., 2009), Lepper et al. chose an elegant approach to critically examine the ‘differentiation recapitulates development’ paradigm. They analyzed the role of Pax7 in pre-adult and adult tissues, a topic on which there have been contradicting conclusions. Pax7 is a transcription factor that is part of the paired box protein family. The protein contains both a paired box domain and a homeodomain (Schafer et al., 1994). It exhibits high sequence and structural homology with another paired box family protein, Pax3, leading to speculation that the two proteins may share overlapping or compensatory roles (Schafer et al., 1994). During development, Pax7 is expressed in cells in the central region of the dermomyotome following Pax3 expression. Pax3 lack-of-function mice (due to spontaneous or induced inactivating mutations) do not develop limb musculature, which is due to the defect in pre-myoblast cell migration (Messina and Cossu, 2009). In contrast, Pax7-null mice develop normal skeletal muscle during embryonic development and display lack of muscle repair in neonatal animals (Seale, 2000). It is thought that the majority of dermomyotome-derived muscle progenitor cells go on to differentiate into skeletal muscle whereas the rest remain in a less differentiated state, serving as a reserve stem cell population in adults (Messina and Cossu, 2009). Notably, during post-natal muscle growth, embryonically-derived myoblasts persist in the satellite cell position and contribute to the rapid formation of myofibers in neonatal and juvenile animals (Ontell and Dunn, 1978).
Pax7 was identified by Seale et al. (2000) to be a marker of muscle stem cells (satellite cells). Unlike Pax3-null mice, some Pax7-null mice are not embryonically lethal, and some strains (129 sv/J background) survive till adulthood. However, they lack muscle stem cells (satellite cells) and exhibit increased rates of muscle degeneration and impaired muscle regeneration compared with wild-type littermates (Seale et al., 2000). Published results regarding the biological function of Pax7 have been contradictory. McKinnell et al. reported that Pax7 promotes myogenesis by indirectly upregulating the myogenic gene Myf5. It purportedly does this by recruiting the Wdr5–Ash2L–MLL2 histone methyltransferase complex, which methylates lysine 4 on histone H3, thereby leading to the opening of the surrounding chromatin (McKinnell et al., 2008). However, Olguin et al. (2007) reported that Pax7 inhibits myogenesis by impeding the function of another myogenic gene, MyoD.
Lepper et al. generated two novel alleles for Pax7. For the first allele, designated Pax7f, loxP sites flank exon 2 of the Pax7 gene. In the second allele, designated Pax7CE, the Pax7 promoter drives the expression of a tamoxifen-inducible Cre recombinase/estrogen receptor (CreER) fusion protein. In animals carrying both alleles, the second exon of Pax7 will be deleted upon tamoxifen induction in all cells in which the Pax7 promoter was active at that particular point, leading to a frame shift that produces a stop codon in exon 3. Although a small peptide may still be translated, it is unlikely that it will retain the biological activity of the wild type peptide, especially as exon 3 codes for part of the homeodomain. The major advantage of this approach over previous ones is that it allows the authors to precisely control the onset of Pax7 gene inactivation, allowing them to study the effects of inactivating the gene at different time points in an animal's lifespan. For a summary of the experimental outline, refer to Figure 1A.
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. Success in muscle regeneration and satellite cell responses after injury by cardiotoxin was compared between Pax7-expressing and Pax7-mutated cells with active Pax7 promoter. (B) Mice heterogeneous for the wild-type and CreER alleles were crossed with Rosa26 reporter mice, which express β-galactosidase (β-gal) in the presence of Cre activity. All regenerating fibers were positive for β-gal expression in this strain and in mice homozygous for the Pax7CE allele. By inducing Cre expression in mice of different ages, it was found that Pax7 expression was vital to myogenesis in neonatal and juvenile mice, particularly those of or younger than P21, but not in adult mice.One caveat regarding this method is that the authors do not prove that tamoxifen induction in Pax7f/Pax7CE effectively destroys the biological activity of Pax7 protein. Pax7 is known to have alternative splice forms. It is possible, albeit unlikely, that deletion of exon 2 in Pax7 could affect splicing of the pre-mRNA, altering or even partially restoring the biological activity of the final protein. Demonstrating that the expression of Pax7-downsteam genes changes in treated vs. control mice would provide more conclusive evidence that Pax7 is effectively inactivated in the treated population.
The authors do show that compared with untreated Pax7f/Pax7CE, wild-type Pax7 protein is no longer detectable by western blot in tamoxifen-treated mice. However, for their western blots, the authors fail to definitively show that they are in fact detecting Pax7 from satellite cells. They probe for Pax7 protein expression in the tibialis anterior muscle without purifying satellite cells from the muscle. Since the acutely injured muscle used in the study certainly has numerous differentiated myoblasts, the detection of Pax7 protein in control mice and down-modulation in tamoxifen-treated mice were not convincingly linked to muscle stem cells. IgG1 contamination in the sample suggests the presence of infiltrating immune cells, and it is quite likely that in addition to differentiated myoblasts known to express Pax7, many other cell types are also present in the lysate. Also, the ability to detect Pax7 in whole muscle without employing techniques such as immunoprecipitation is remarkable given that satellite cell proteins represent a small fraction of the total protein present in muscle, and in conjunction with the lack of data on pure populations of quiescent satellite cells from uninjured muscle this opens doubts as to whether Pax7 protein was indeed lacking in this subset of muscle stem cells.
Contrary to published results using Pax7-null mice (Seale et al., 2000), the authors found that selectively deleting the Pax7 gene in adult mice did not lead to satellite cell attrition nor diminish the ability of satellite cells to repair muscle following cardiotoxin-induced injury. By inducing Cre expression in mice of different ages, they found that Pax7 expression was vital to myogenesis in neonatal and juvenile mice, particularly those of or younger than postnatal day 21 (P21, Figure 1B). Mice treated with tamoxifen and subjected to cardiotoxin injury at this age had significantly fewer de novo myofibers, which are easily identified via histology by their centrally-located nuclei, compared with untreated mice. The authors concluded that P21 marked the transition of satellite cells from a proliferative state to a quiescent state. Once again, it needs to be noted that neonatal and juvenile muscle contain differentiated fusion-competent myoblasts which express Pax7 protein. However, these myoblasts, unlike satellite cells, do not return to quiescence and are present in satellite cell position only transiently (before fusing into new myofibers). Therefore, the relevance of Pax7 in myoblasts, but not necessarily in muscle stem cells was addressed in P0–P21 animals. Once the juvenile proliferative stage of muscle growth is completed, the dramatic shift in muscle mass and strength occurs before mice reach sexual maturity, which is at 
P42 for females and P56 for males. Characterizing the mechanism behind this marked transition in muscle stem cell function could provide valuable insight into stem cell biology, with implications for stem cell function relating to aging and possibly even oncology.
The authors assert that descendants of Pax7 positive cells are a major source of regenerating myofibers. The method used to reach this conclusion involved crossing mice heterogeneous for the wild-type and CreER alleles with Rosa26 reporter mice, which express β-galactosidase (β-gal) in the presence of Cre activity (Figure 1B). All regenerating fibers were found to be positive for β-gal expression. In addition, all regenerating fibers from surviving mice homozygous for the Pax7–CreER allele were also positive for β-gal expression. These data are in line with previously published findings, demonstrating that the Pax7 promoter is active in myogenic lineage cells of adult mice.
Of special significance is the finding that inactivating Pax7 in adult muscle does not lead to any noticeable changes in muscle phenotype. Following tamoxifen induction, cardiotoxin injury and a period of regeneration, cells negative for Pax7 but positive for β-gal, indicating that they once expressed Pax7 mRNA, and positive for the adhesion protein M-Cad that is expressed by satellite cells and myoblasts, were found in the satellite cell position outside of the myofiber plasma membrane but beneath the basal lamina. After a second round of injury, mice treated with tamoxifen exhibited the same robustness of muscle regeneration as control mice. The authors interpret this finding as indicative of the regeneration capacity of these Pax7-negative satellite cells. However, they do not show that these cells are indeed the ones which contribute to new myofiber formation. They themselves note that it is impossible to rule out the potential role of infiltrating cells in compensating for Pax7 depletion; it has been reported that bone marrow cells may also contribute to myofiber formation (LaBarge and Blau, 2002).
Although Pax7 does not seem to be critical for adult myogenesis, the study does not necessarily indicate that Pax7 does not play a role in adult myogenesis. Perhaps Pax7 affects some other aspect of myogenesis not directly addressed by the injury-regeneration model. Even if absolutely no phenotype could be found, Pax7 could still be playing a role in myogenesis. In mammalian systems, signaling pathways are often redundant, with several homologs of the same protein present. Distinct signaling pathways can also have overlapping targets (Kafri et al., 2009). Although Pax3 does not seem to compensate for Pax7 activity, it is possible that other related proteins or even completely separate signaling networks could compensate for Pax7 in the adult but not in the embryo or the pups. Deleting Pax7 alone will not disrupt these compensatory mechanisms, and hence may not result in a distinct phenotype.
Overall, the fundamental significance of Lepper et al.'s work is in demonstrating that there are phenotypic differences in juvenile and adult muscle with respect to Pax7 depletion, forcing the stem cell field to rethink a common approach in studying adult stem cell populations. Although at present there is no evidence that their results may be extendable to other cell and tissue types or even for normally regenerating muscle that has not been subject to acute trauma, this study shows differences in the biology of developing and developed tissues, prompting more caution in extrapolating embryonic and developmental data to adult systems.
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