In recent years, several new markers of quiescent satellite cells have been identified, providing insight into the origins of these cells as well as the mechanisms by which they affect muscle regeneration. Using RDA, we have identified several potential satellite cell markers, including Megf10, a multiple EGF repeat–containing transmembrane protein (Seale et al., 2004
). Although orthologues of Megf10 (CED-1 and Draper) have been previously identified and shown to function in engulfment of apoptotic cells during neurogenesis, a role for Megf10 in satellite cell function has not been previously described (Zhou et al., 2001
; Freeman et al., 2003
Megf10, although highly expressed in the brain, is also expressed in resting adult skeletal muscle, as determined by in situ hybridization, and its expression is up-regulated in response to injury as determined by quantitative PCR. Our immunohistochemical analysis on sections from tibialis anterior muscles and individual fibers isolated from EDL muscle clearly demonstrate that the expression of Megf10 is strictly limited to the majority of quiescent satellite cells in resting muscle. Although we only detected Megf10 in Myf5-Cre-YFP+/Pax7+ satellite cells, it is possible that Megf10 expression level in the quiescent Myf5-Cre-YFP−/Pax7+ satellite stem cell population is below detectable levels in our assays. Interestingly, we observed Megf10 expression in all satellite cells of individual fibers after activation, suggesting that all satellite cells up-regulate Megf10 upon activation. This is in agreement with our quantitative PCR analysis, which demonstrated significant up- regulation of Megf10 in activated satellite cells.
Our results demonstrate that overexpression of Megf10 in myogenic cells in vitro results in enhanced proliferation. In wild-type myoblasts, Megf10
expression is down-regulated during terminal differentiation. In cells modified to overexpress Megf10, expression is maintained after serum withdrawal and a dramatic inhibition of differentiation is observed. Importantly, cells overexpressing Megf10 exit the cell cycle within 24 h of serum withdrawal, as determined by BrdU incorporation, but maintain the ability to reenter the cell cycle through serum stimulation. These results suggest that maintained Megf10 expression allows cells to return to a quiescent state where they can respond to subsequent stimulation. Collectively, these data implicate Megf10 as being an important regulator of satellite cell function by suppressing the progression of the differentiation program of sublaminar satellite cells and thus enforcing self-renewal. It has recently been demonstrated that in the absence of Myf5, the transient activating population is dramatically compromised in adult skeletal muscle, and in response to injury, activated satellite cells undergo precocious differentiation in viable Myf5−/−
mice (Ustanina et al., 2007
). Thus, it would appear that although MyoD expression is required for the appropriate differentiation of activated satellite cells, Myf5 functions in the expansion and proliferation of these cells. Interestingly, cells overexpressing Megf10 express elevated levels of Myf5 and decreased levels of MyoD, much like MyoD−/−
myoblasts, which show a propensity for proliferation and self-renewal rather than differentiation (Megeney et al., 1996
; Sabourin et al., 1999
). Although there is an increase in the level of Myf5
RNA in these cells, it has not been determined if this is a direct effect of Megf10 overexpression stimulating Myf5
transcription. Alternatively, repression of the Myf5
locus may be relieved because of the absence of MyoD protein (Megeney et al., 1996
). We believe the latter to be the case, as knockdown of Megf10
expression in MyoD−/−
myoblasts does not result in down-regulation of Myf5
. In fact, Myf5
transcript levels increase upon Megf10
silencing. Interestingly, we have previously demonstrated that Myf5 levels are elevated at early time points after serum withdrawal in MyoD−/−
myoblasts (Sabourin et al., 1999
). Based on our results, one could speculate that Megf10 may suppress the progression of the differentiation program of sublaminar satellite cells by modulating Myf5 and MyoD expression.
The role of Megf10 in regulating the proliferative potential of the satellite cell compartment is further supported by our knockdown experiments in vitro and on individual fibers. In vitro, knockdown of Megf10 resulted in precocious differentiation of primary myoblasts maintained under growth conditions. Megf10 knockdown on individual fibers resulted in a pronounced decrease in the number of activated precursor cells because of precocious differentiation, as determined by a significant increase in the number of myogenin-expressing cells at 60 h, a time at which the number of cells per fiber (si-Megf10 vs. si-scrambled) is not significantly different. Megf10 knockdown also resulted in a concomitant reduction in the number of transient self-renewing cells per fiber after 72 h in culture. Furthermore, it is important to note that the overall proportion of Myf5-Cre-YFP− stem cells to Myf5-Cre-YFP+ committed progenitor cells was unaltered, implying that loss of Megf10 expression results in precocious differentiation of both the stem and progenitor satellite cell populations. This is not unexpected, given that after activation all satellite cells express Megf10. These results imply a critical role for Megf10 in the appropriate self-renewal of the satellite cell compartment.
It has been well demonstrated that Notch signaling is a critical determinant in the progression of satellite cells toward myogenesis (Conboy and Rando, 2002
). Satellite cell activation and proliferation is accompanied by an increase in expression of Notch receptors and Notch signaling, whereas terminal differentiation occurs after inhibition of Notch signaling (Conboy and Rando, 2002
). All satellite cells express Notch-1 and satellite stem cells (YFP−
) express Notch-3
, whereas the committed progenitor population (YFP+
) expresses the Notch ligand Delta-like-1
(Kuang et al., 2007
). Inhibition of Notch signaling results in a loss of the stem cell population and premature differentiation, suggesting that the Notch signaling pathway plays a crucial role in the self-renewal of satellite cells (Kuang et al., 2007
Of interest is the potential interaction of Megf10 with the Notch signaling pathway. A recent publication demonstrates the ability of JEDI, a potential Megf10 family member, to regulate the Notch signaling pathway in a delta-like manner in hematopoietic cells (Krivtsov et al., 2007
). Furthermore, it has been demonstrated that constitutive activation of Notch signaling in myoblasts results in enhanced proliferation and defective differentiation, a phenotype that is strikingly similar to that observed upon overexpression of Megf10 in myoblasts (Nofziger et al., 1999
; Conboy and Rando, 2002
Our results suggest that Megf10 regulates the proliferative capacity of myogenic cells, potentially via the Notch signaling pathway. With 30% transfection efficiency, we were able to reduce Megf10 expression 0.56-fold overall in primary MyoD−/− myoblasts. This was accompanied by a reduction of Notch receptors' transcripts and down-regulation of Hes1, which can be directly linked to the precocious differentiation observed in wild-type myoblasts after Megf10 knockdown, a result reminiscent of a loss of Notch signaling. Up-regulation of Myf5 after Megf10 silencing in MyoD−/− myoblasts, coupled with the observed decrease in Notch and Hes1 transcript levels, suggests that down-regulation of Megf10 allows cells to progress toward terminal differentiation. However, Megf10 silencing did not rescue the differentiation deficit observed in MyoD−/− myoblasts (unpublished data), most likely because of the requirement for MyoD activity for subsequent differentiation.
It is curious that we only detected Megf10 expression in the committed progenitor population of quiescent satellite cells, which also express Delta-like-1, whereas it was up-regulated in all satellite cells after activation. Such heterogeneity within the quiescent satellite cell compartment has previously been described for other markers such as CD34 and Myf5
as well as components of the Notch signaling pathway and may be indicative of the hierarchical nature of the satellite cell compartment (Beauchamp et al., 2000
; Conboy et al., 2003
; Kuang et al., 2007
). Alternatively, the heterogeneity observed within the satellite cell compartment may reflect differences between naive satellite cells established during development and those that have been activated subsequent to their developmental establishment. Importantly, these concepts are not mutually exclusive.
Given the ability of Megf10 expression to alter growth and differentiation kinetics of myoblasts as well as the levels of key myogenic proteins, we hypothesize that Megf10 functions to maintain the satellite cell compartment. It does so by maintaining self-renewing cells' proliferation and by inhibiting their progression through the myogenic program and terminal differentiation. After activation of satellite cells, Megf10 may dictate whether a cell continues to proliferate, returns to a quiescent state, or enters the myogenic program to undergo terminal differentiation. This, we propose, is accomplished by modulating Notch signaling. Although the mechanism by which Megf10 expression is regulated remains to be elucidated, our findings demonstrate a novel and critical role for Megf10 in the regulation of satellite cell dynamics and maintenance.