Research
The health of postnatal animals is dependent on the ability of adult stem cells to respond to environmental cues and adapt accordingly. By definition, stem cells have the ability to self-renew, as well as differentiating to repair/maintain tissue integrity. How do they decide what to do?
In our laboratory, we use muscle stem cells (called satellite cells, because of their localisation) as a paradigm for the study of adult stem behavior.
Satellite cells provide an excellent model because they can be studied bothin vivo(for example using knockout and transgenic mice and the Cre-Lox system) and in vitro, following isolation. In particular, we are interested in the signaling mechanisms that dictate satellite cell fate decisions, ranging from extracellular stimuli (such as growth factors) to chromatin structure and subsequent gene expression (e.g. by epigenetic modifications).
Even though adult myogenesis is initiated in a different manner from embryonic myogenesis, the major cellular processes are parallel: cells may migrate (after injury), upregulate the myogenic determination transcription factors (MDFs) Myf5/MyoD, undergo a period of regulated proliferation to generate progenitor cells before upregulating the MDF myogenin, exit the cell cycle, express muscle-specific structural proteins and finally fuse to form hypertrophied/replacement multi-nucleated myofibres. In addition to differentiation, adult muscle stem cells must also self-renew; this is subject to controversy but could be via a combination of asymmetric and symmetric cell divisions, as well as dedifferentiation of precursor myoblasts.
Part of our focus is on the signalling pathways that regulate the transition from proliferation to irreversible differentiation. We and others have demonstrated the importance of both PI3K/Akt and p38 MAPK in myoblast differentiation. We have further shown that p38 is activated earlier than Akt in the myogenic programme. Even though upstream activators for myogenic PI3K/Akt have long been identified (principally via activation of receptor tyrosine kinases, e.g. IGF1R), upstream mechanisms by which p38 is activated were unknown until recently.
We demonstrated that cell-cell interactions, via cadherins, are pivotal in determining p38 activation in myogenesis, and this principle has been supported by others investigating alternative mechanisms of signalling cell-cell interactions (e.g. Cdo). We also revealed that p38 has an important role in communicating the cellular environment to the nucleus, upregulating the expression of pro-myogenic agents, such as IGF-II and even Akt2, thus providing a feed-forward mechanism to propel myoblasts though the differentiation programme.
As the satellite cell occupies a specific spatial niche between the basal lamina and the myofibre membrane, it can in theory receive specific and different signals from the basal lamina and the myofibre membrane, as well as via soluble molecules that form part of the cell’s milieu. Access to these signals could dictate cell fate decisions.
The extensive genomic reprogramming that must occur during satellite cell activation, proliferation and differentiation is established via changes in chromatin structure that make it permissive for transcription or silencing at specific loci. The compaction of chromatin is pivotal in determining DNA accessibility of transcriptional machinery, and this is in part regulated by post-translational modifications of histone proteins i.e. by epigenetic mechanisms. For example, the chromatin of actively expressed pro-myogenic genes will be in an open conformation in late differentiation whereas that for proliferation genes will have been silenced.
Trithorax (TrxG) mediated histone 3 lysine 4 trimethylation (H3K4me3) is associated with genes marked for expression; polycomb repressive complex 2 (PRC2) mediated histone 3 lysine 27 trimethylation (H3K27me3) is associated with inaccessible chromatin and repression/silencing. Even though further important epigenetic mechanisms regulate chromatin structure, the methylation of H3K4 and H3K27 are thought to form initial and reversible marks, on the basis of which subsequent modification occurs e.g. DNA methylation. Both PRC2 and TrxG exist as multi-protein complexes that are regulated by post-translational modifications, and therefore intracellular signalling pathways have the potential to dictate their activity.
Babraham is fortunate to have two Illumina next-generation sequencers, by which we can perform genome wide sequencing e.g. ChIP-Seq to investigate global epigenetic and gene expression regulation in muscle stem cells.
In addition to muscle cell lines (which are useful but have limitations), we can isolate muscle satellite cells ex vivo, allowing us to use genetically modified mice to examine the interactions between cell signalling cascades and chromatin structure. Differentiation of wild-type adult muscle stem cells is shown below:
Updated 23 August, 2011