Genetic and cellular regulation of stem to differentiated states in mouse skeletal muscle – Tajbakhsh

Scientific background and objectives: Stem cells are crucial for establishing and maintaining tissues and organs. Only rudimentary information is available on stem cell self-renewal and differentiation. This proposal aims to characterise stem cells and their daughters in skeletal muscle. One important aspect of this work, relies on elucidating the lineage and the stem cell niche. Work from a number of laboratories including our own, has shown that skeletal muscle identity is acquired from muscle stem cells by the expression of the myogenic factor genes:Myf5, MyoD or Mrf4. Unique genetic tools generated in the laboratory have permitted us to identify a novel population of stem cells in this tissue. Triple mutant mice totally lack skeletal muscle, however, the stem and progenitor cells can be tracked using genetic markers.In addition Pax3 and Pax7 are important for stem/progenitor cell specification. Therefore Pax3/7 and the myogenic factors play critical roles in establishing and driving muscle identity from stem cells. Using reporter genes targeted to the Pax7 and Myf5 loci we can track these stem cells and their daughters. We have used several strategies to characterise the stem to differentiated transitions, and are focusing particular attention on the regulation and maintenance of stem and progenitor cells.

Description of project, methodology: One objective is to describe the cell lineage for skeletal muscle. We have proposed a working model defining stem, progenitor, precursor and differentiated cell states during embryonic skeletal muscle development. Using this framework, we have generated Pax7EGFP/nlacZ reporter mice where nlacZ reporter gene expression is blocked by the presence of the "stop cassette" EGFP. Cre gene expression, introduced by genetic crosses, mediates removal of the "stop cassette" and results in nlacZ gene expression. These mice will be crossed to appropriate inducible Cre expressing lines and the derivatives of the cells can be followed restrospectively in clones in mosaic embryos. Complementary to these and other related genetic strategies, we are examining the onset of expression of Pax3, Pax7, Myf5, MyoD and Mrf4 using antibodies against the native proteins, or reporter genes in knock-in mice. This approach will provide important spatiotemporal information necessary for assessing sibling relationships in the lineage. The localisation of stem cells with Pax3 and Pax7 as markers permits us to assess their topological positioning in the muscle masses. Our question here is whether extrinsic or intrinsic signals, or both, are required in preventing the total stem cell population from differentiating. First, we will determine if the stem cells are in different "niche" locations with respect to committed myoblasts. Candidate neighboring cells include endothelial cells, vessels and connective tissue. In addition, we will assess whether downstream components of the signalling pathways such as Wnt, BMP, and Notch, are acting selectively in these cells, possibly influencing their self renewal. These studies will be carried out with specific antibody reagents for these pathways. Another strategy involves investigating adult muscle stem cells. During postnatal development, satellite cells are the principal regenerative cell type. We have shown recently that these cells possess stem cell character. Specifically, pulse-chase labelling studies with BrdU in vivo showed that a sub-population of cells are label retaining. Importantly, we demonstrated that during cell division, the label segregates to only one daughter cell. This observation is consistent with the model proposed by Cairns that stem cells retain selectively old "immortal" DNA templates. Importantly, we have shown that the general cell asymmetry apparatus monitored by segregation of the cell fate determinant Numb is linked to template DNA segregation. We have initiated siRNA studies in isolated satellite cells to perturb Numb function and assess its role in cell asymmetry and self-renewal. Live cell imaging with EGFP-fusion constructs are an integral part of this programme to assess the cell behaviour of adult muscle stem cells in vitro and in the organism and investigate the mechanisms.

Expected results: Our developmental approach has proven to be informative in identifying and characterising stem and progenitor cells in skeletal muscle. This proposal is mostly fundamental in nature and geared to identify the molecules which promote their self-renewal. Their characterisation and regenerative potential can subsequently be tested in animal models for long term grafting in cell and genetic therapeutic contexts. We believe that a systematic investigation of the biology of skeletal muscle stem cells is an needed before therapies in humans can be seriously attempted. More generally, investigations on normal stem cell regulation should also provide insights into how deregulation can provoke the disease or the cancer states.