Chapter 5
The work presented in this thesis addresses many of the general questions regarding the nature and function of satellite cells as adult myoblasts presented in Chapter 1. The results clearly indicate that while satellite cells may resemble myoblasts of the embryo in some aspects, they differ in both their gene expression and gene usage, leading to alternate pathways for some common functions and at least partially explaining the mechanisms of some functions unique to satellite cells.
In the embryo, cells become committed myoblasts and proceed to differentiate in a
continuous and fairly rapid process. In contrast, satellite cells are presumably committed to the myogenic lineage but do not differentiate until long after embryogenesis, and then only when the host muscle tissue is damaged. While it remains unknown how either embryonic myoblasts or satellite cells become committed to the myogenic lineage, work presented here presents a mechanism by which committed but quiescent satellite cells become activated to differentiate. The hepatocyte growth factor/scatter factor receptor c- met, which is demonstrated in Chapter 2 to be expressed by quiescent satellite cells in vivo, is an excellent candidate for the transducer of the initial activation signal. This argument is supported by the biochemical similarity of the c-met ligand to the major active component in crushed muscle extract (Bischoff, 1986) and the ability of exogenous HOF/SF to induce activation in rat satellite cells in vitro (Allen et aI., 1995) and in vivo (Tatsumi et al., 1998). Additional functions of c-met in satellite cells, such as inducing cell migration (Bischoff, 1997), may also be important for robust regeneration.
During embryonic myogenesis, the myogenic regulatory factors (MRFs) play partially redundant roles in myogenic determination and differentiation. In particular, MyoD and myf5 share a function in determination of somitic myoblasts: deletion of either gene from the germline does not result in a myogenic defect due to compensation by cells expressing the other factor (Rudnicki et al., 1992), but deletion of both leads to the absence of myoblasts (Rudnicki et
at.,
1993). MRF expression is crucial for all known skeletal myogenesis, and satellite cells were known to express MRFs duringdifferentiation, so it was extremely likely that MRF activity also played a pivotal role in satellite cell myogenesis. However, when this work was begun the roles of specific MRFs in satellite myogenesis had yet to be defined; since adult muscles and potentially their satellite cells are heterogeneous (i.e. derived from axial vs. appendicular myoblasts, or expressing fast vs. slow myosin heavy chain isoforms) there also existed the possibility of differential gene expression within the satellite cell population. Possibilities for MRF activity in satellite cells included a mechanism similar to that of embryonic myoblasts, in which either MyoD or myf5 is directly required for initiation of myogenesis in different populations of satellite cells; one in which expression of either MyoD or myf5 was sufficient for any satellite cell; one in which either MyoD or myf5 was required for all satellite cells; or a completely different mechanism requiring, for example, myogenin expression.
In Chapter 2, it was determined that primary satellite cell in culture first expressed
either MyoD or myf5, followed shortly by coexpression of both. This differed from the temporal coexpression seen in either embryonic myoblast population, and indicated that at least a mechanism for initiation of myogenesis in satellite cells which involved neither MyoD nor myf5 was unlikely. In Chapter 4, building on in vivo morphological studies published while this work was in progress (Megeney et ai., 1996), it was determined that MyoD is specifically required for robust myogenesis in satellite cells. That satellite cells unable to express MyoD also failed to express MRF4 at later points in the response and that very few MyoD-null satellite cells ever expressed m-cadherin are probably symptomatic of the failure of myogenic progression in these cells as well as being themselves causes of certain aspects of the differentiation-defective phenotype.
The broader examination of genes whose activity may affect processes such as proliferation and differentiation, and the balance between them, presented in Chapter 3 was meant to suggest sets of genes for later coexpression analysis in single cells. Many of these genes and gene families were chosen for study because they are known to
influence myogenic development in the embryo. It is important to realize, however, that the extracellular environments in which satellite cells exist before and after muscle damage are different from each other as well as from the somitic system; therefore, it is unlikely that all genes known to affect embryonic myogenesis will be expressed by satellite cells, or that genes which are expressed will necessarily serve the same function.
Within gene families, it was hoped that a preferred suite of genes would be expressed in satellite cells, thus reducing the complexity of the system. While within some families most or all members were found expressed in satellite cells (i.e. the MRFs, MEFs and Ids), in other families (i.e. Notches and Dlls) there were some family members which were never detected in satellite cells. Given the results presented in Chapter 3, several sets of genes whose coexpression patterns in single cells would be of interest suggest themselves; following are a few possible sets.
Since the MEF2 family of transcription factors are thought to act synergistically with MRFs to activate muscle-specific genes, determining the fractional representation of MEF2A, C, and D and any possible coexpression preferences with MRFs and correlation to the differentiated state may suggest specific roles for each factor in satellite myogenesis. Fractional representation and coexpression with MRFs should also be determined for Ids 1-4; if there appears to be an ordered progression of MEFs and Ids during the course of differentiation, it may also be possible to correlate sets of MRFs, MEFs, and Ids in order to form a more complete picture of the myogenesis-promoting and -inhibiting factors at work within a single satellite cell.
Similarly, the coexpression of genes in the Notch cell-cell signaling pathway (including Notches 1 and 2, Dll-l, Jagged-2 and possibly Jagged-I) with the MRFs is also of interest, especially in adjacent satellite cells after 48 hours, which are likely to be siblings. It may be possible to determine specific roles for these signaling molecules in promoting or inhibiting the differentiated state. Their coexpression with Radical and
Lunatic Fringe and numb and numblike, especially if these genes are indeed differentially expressed based on a given cell's terminal differentiation status, is also of interest.
The pathology of MyoD-null satellite cells in culture offers several opportunities for studying processes which do not occur in wild-type cells in vitro, such as a potential to return to a state similar to quiescence. The analysis of genes specifically expressed in apparently regressing cells may yield clues as to the mechanism of return to quiescence.
The first such differentially-expressed gene, Msx-l, is expressed in quiescent satellite cells in both wild-type and MyoD-null mice, thus allowing protein and RNA reagents to be characterized in sections of intact muscle before being applied to sections of damaged muscle or to cultured fibers in order to confirm and extend the pool RT-PCR data.
In conclusion, this work presents significant technical advances in the study of satellite cells, significant new data based on these techniques, and suggests new lines of questioning which have the potential to further extend knowledge of this system.