Given the heterogeneity described in cells associated with myogenesis, it has been difficult to identify specific markers that are unique to the satellite
cell lineage. MyoD plays an important role in muscle differentiation, and yet its loss does not prevent muscle formation, therefore a recent screen was performed to try to identify novel specific markers for MyoD dependent muscle differentiation. Myoblasts isolated from wildtype and from MyoD-/- mice were used in the screen, and differences in RNA expression were analysed. The discovery of 51 differentially expressed genes were discovered, and 40 of these were analysed by western blotting in myoblasts and differentiated cells, for both the wildtype and MyoD knockout. This screen identified MD p67, the mouse homolog of human MEGF10 (XM_140362), as expressed in proliferating wildtype and MyoD-/- myoblasts, but not in differentiated cells, and that MyoD knockout myoblasts expressed slightly higher levels of Megf10 than wildtype cells, although it was not selected as an example of a gene with significantly higher expression in knockout cells (Seale et al., 2004).
This work was then followed up by a focused study on MEGF10 in satellite cells, confirming by qPCR that Megf10 was down regulated at the mRNA level by ~6.4 fold when primary myoblasts, isolated from wild-type mice (as described previously (Megeney et al., 1996; Sabourin et al., 1999)) terminally differentiated, and that MEGF10 expression was ~1.4 fold higher in MyoD-/- myoblasts, isolated from MyoD-/- mice (Ishibashi et al., 2005) ,
compared to wildtype myoblasts, and was down regulated by ~8 fold after +, CD31 /Sca1 /CD45 satellite cells, 94% positive for Pax7, showed ~100 fold upregulation of MEGF10 in satellite cells isolated from 8 week old mice that were activated by in vitro culture for three days compared to freshly isolated satellite cells. In situ hybridisation with an RNA probe also showed Megf10 expression in cells along the edges of muscle fibres in tibialis anterior (TA) muscle fibres, a similar localization to satellite cells. Finally, using an antibody raised to the cytoplasmic domain of MEGF10, this study also showed that MEGF10 was only expressed in Pax7 positive satellite cells (Holterman et al., 2007).
The authors then went on to express an HA-tagged full length MEGF10 protein construct in C2C12 cells, which was shown by western blotting to have a molecular weight of ~120 kDa, in which they probed with the anti-HA
antibody but not directly with an anti-MEGF10 antibody. Based on its sequence MEGF10 is predicted to have a molecular weight of 122 kDa, similar to that seen by western blot. However, this would not include any increase in size due to PTMs, which might be expected given that the majority of the protein is extracellular and primarily composed of EGF-like domains, which are typically heavily glycosylated (1.3.3). Moreover, the antibody raised against the C-terminal 290 aa of MEGF10 was only used in immunostaining without any independent characterisation of the antibody to show that it recognised the expressed HA-tagged protein either by western blotting or by immunofluorescence. Overexpression of HA-tagged MEGF10 in C2C12 cells (but not fibroblasts) slightly increased growth rates, and markedly decreased the ability of the myoblasts to fuse and differentiate into myotubes, as well as down regulating MyoD expression potentially returning the cells to a quiescent state. Using siRNA to knockdown MEGF10 expression in satellite cells on cultured muscle fibres promoted precocious activation of satellite cells, as evidenced by an increase in myogenin expression and increased differentiation in cultures of primary satellite cells. Lastly, knocking down MEGF10 by 0.56 fold in cultured MyoD-/- cells reduced expression of Notch 1, Notch 2 and Notch 3, and it was suggested that MEGF10 might be able to activate Notch signalling. Notch, like MEGF10, is a TM protein that contains several EGF-like repeats in its ECD and is activated by interaction with the DSL ligands of proteins such as Jagged. However, MEGF10 does not contain any DSL repeats, and therefore would have to interact with Notch via a different mechanism (Holterman et al., 2007).
These experiments led to the hypothesis that MEGF10 has a role in regulating proliferation in myogenic cells through the Notch signaling pathway. Notch has been shown to be critical in facilitating the progression of satellite cells towards myogenesis (Conboy and Rando, 2006), and is therefore important in development. Blocking of Notch signalling causes premature differentiation of myogenic progenitors, and formation of very small muscle groups, a similar phenotype to that found in EMARDD patients (Schuster-Gossler et al., 2007; Vasyutina et al., 2007). During the perinatal period in mice, satellite cells continue to proliferate until postnatal day 21,
when they adopt quiescent characteristics (White et al., 2010). This is dependent on Notch signalling, as well as target genes, Hey1, HeyI and Sprouty1, which negatively regulate tyrosine kinase signalling (Bjornson et al., 2012). Like MEGF10, activated Notch signalling suppresses differentiation of cultured C2C12 cells, suggesting a critical role for MEGF10 in maintaining the satellite cell compartment through self-renewal, possibly in a similar manner to Notch. Mutation of the Notch ligand, DLL1, or knockdown of Rbpj, the DNA binding protein, which regulates Notch expression, results in early depletion of the myogenic progenitor pool, and also results in the formation of small muscle groups that do not contain any satellite cells (Vasyutina et al., 2007; Bjornson et al., 2012). During foetal development the knockdown of Rbpj is accompanied by rapid MyoD upregulation, but ablation of MyoD does not properly rescue satellite cell depletion. Instead these cells do not migrate normally, and Pax3+ cells are found instead in the interstitial space, and not the basal lamina, suggesting that Notch is important for regulating the formation of basal lamina around satellite cells. Therefore, activated, emerging satellite cells show disruption in expression of basal lamina components and adhesion molecules including MEGF10 through its interaction with Notch (Brohl et al., 2012).
This study also assessed the changes to MEGF10 expression in coRbpj: MyoD-/- (RM) and MyoD-/- mice compared to control mice. In contrast to the earlier report that expression of MEGF10 is increased in MyoD-/- mice, (Seale 2004), here it was reported that MEGF10 shows a 1.8 fold decrease in MyoD-/- mice, which though not significant, rose to a significant 3.1 fold decrease in the RM mice. Furthermore, induction of MEGF10 expression was shown to be dependent on DLL1, shown by a significant increase in the levels of MEGF10 in the presence of DLL1. Finally, it was reported that low levels of MEGF10 were observed in the plasma membrane of Pax3+ cells in the RM mice, at embryonic days 15.5 and 17.5, using a commercial antibody (Santa Cruz), raised to the cytoplasmic domain of MEGF10 (Brohl et al., 2012).
Although these studies identified a possible function of MEGF10 in myogenic proliferation evidenced by a link between MyoD and MEGF10, myogenin has
also been identified as a positive regulator of MEGF10 transcription in skeletal muscle. By analysing the 5’ end of the Megf10 gene, a myogenin binding motif was identified that was shown to bind to myogenin (Park et al., 2014). Moreover, levels of MEGF10 and myogenin expression rose in ctx damaged muscle as measured by qPCR. A luciferase reporter assay that showed the activity of the MEGF10 promoter, showed an 11 fold increase in expression resulting from its interaction with myogenin, whilst Myf5 had a smaller effect increasing MEGF10 promoter activity by only 2.8 fold. Myogenin was shown to bind directly to this region of DNA and its effect on MEGF10 promoter activity is dose dependent (Park et al., 2014). However it is unclear how MEGF10 can promote proliferation, but inhibit differentiation as reported (Holterman et al., 2007), if it interacts with myogenin, which is not expressed until the cells begin to terminally differentiated, and would not be expected to affect proliferation.
Overall, these studies suggest that MEGF10 has a clear role in neuronal cells, where it is in involved in the clearance of apoptotic cells (Iram et al., 2016) as well in synapse elimination (Chung et al., 2013) by astrocytes. Both observations are consistent with the role of MEGF10 in patterning retinal neurons (Kay et al., 2012), and the observed effects of knocking out the ortholog of MEGF10 on the brain and retina in Drosophila (Draper et al., 2014).
However, EMARDD patients do not have a neurological phenotype, but do have a muscle deficiency, and it therefore seems likely that MEGF10 is also important in satellite cells. It has been suggested to be important for renewing the satellite cell pool, by maintaining proliferation in cells and inhibiting the progression of the myogenic program in combination with MyoD and Notch (Holterman et al., 2007; Logan et al., 2011). It has also been suggested to promote satellite cell differentiation shown by the relationship between increasing expression levels of MEGF10 and myogenin (Park et al., 2014), although overexpression of MEGF10 inhibits differentiation in vitro (Holterman et al., 2007). MEGF10 has also been suggested to influence the ability of satellite cells to populate the satellite cell niche through interactions with Notch and by promoting the adhesion of satellite cells to the basal lamina or muscle fibre (Brohl et al., 2012). It is difficult to reconcile these disparate ideas and to explain how a mutation in the MEGF10 gene leads to a disruption of the stem cell pool, reduces the ability of skeletal muscle to regenerate leading to the reduced myofibre size seen in EMARDD patients.