Recent work has identified the presence of adult stem cells within most if not all adult tissues that contribute to many tissues following reintroduction in vivo. Our laboratory has identified the paired box transcription factor Pax7 as being required for the specification of myogenic satellite cells from progenitor adult satellite cells. In the absence of Pax7, adult stem cells in muscle appear to undergo default differentiation towards the hematopoietic lineages. In this application, we outline a research program designed to further our understanding of the molecular mechanisms that regulate the lineage commitment of adult stem cells. To elucidate the mechanisms that direct the specification of adult stem cells, we propose to characterize the inductive signal, test candidate factors such as Shh, Wnts, and BMPs, and finally molecularly clone the inducing factors using expression-based screens. Candidate factors with inductive activity will be tested on adult stem cells from different tissues. To investigate whether expression of Pax7 in adult stem cells directly induces their myogenic specification, viral vectors will be used to ectopically express Pax7 in adult stem cells from diverse tissues. The ability of viral vectors expressing Pax7 to induce myogenic specification will be assessed both in vitro and in vivo. Lastly, this approach will be used to elucidate the function of the alternative splice forms of Pax7. To elucidate the broader mechanisms that regulate specification, a PCR-based differential cloning procedure called representational difference analysis (RDA) and microarray analysis will be used to identify potential Pax7 target genes. RDA and microarray analysis will also be used to perform comparative expression profiling between muscle, marrow and neural stem cells. Lastly, microarray profiling and cluster analysis will be performed on adult stem cells undergoing myogenic specification. To investigate the mechanism by which Pax7 enforces lineage commitment, FACS/Hoechst enriched adult stem cells from Pax7-/- mice will be transplanted either intravenously or intramuscularly.
