|Molecular Genetics of Development - URA CNRS 2578|
|HEAD||Prof. BUCKINGHAM Margaret / firstname.lastname@example.org|
|MEMBERS||BODIN Catherine/COQUERAN Sabrina/Dr CRIST Colin Dr DAUBAS Philippe/Dr LAGHA Mounia/Dr LE GARREC Jean-François/LESCROART Fabienne/MARCHISET Sophie/MAYEUF Alicia/Dr MEILHAC Sigolène/Dr MONTARRAS Didier/Dr PALLAFACCHINA Giorgia/ ROCANCOURT Didier/Dr SATO Takahiko/TAISNE Myriam/ Dr VINCENT Stéphane/Dr WATANABE Yusuke
Our Unit works on myogenesis and cardiogenesis, using the tools of mouse molecular genetics to examine cell behaviour and gene function.
We are focussing on skeletal muscle progenitor cells that express Pax3/Pax7. A key question in stem cell biology is how the balance between self-renewal and tissue differentiation is regulated. By identifying targets of Pax3/7 transcriptional regulators in the myogenic context we can provide some answers to this question.
We had previously established that Pax3 and Pax7 play an essential role in assuring the survival and the myogenic potential of muscle progenitor cells. These are derived from the central domain of the dermomyotome of the embryonic somite and are present in all developing muscle masses and also contribute the reserve satellite cells of postnatal muscle.
Our demonstration that the myogenic determination gene, Myf5, is a direct Pax3(7) target, showed how these factors promote the entry of a stem cell into the myogenic programme. We have now identified Fgfr4 as a direct Pax3(7) target. The gene that encodes an intracellular modulator of FGF signaling, Sprouty1, also lies genetically downstream of Pax3 and manipulation of a conditional transgene expressing Sprouty shows how Pax mediated modulation of the FGF signaling pathway can control muscle stem cell renewal versus myogenic differentiation. We are currently investigating the role of Foxc2 which we have shown is negatively regulated by Pax3. The presence of a negative feedback loop between Foxc2/Pax3 in multipotent stem cells in the somite is potentially important in determining myogenic/non-myogenic cell fates.
Our targeting of Pax3 with a GFP fluorescent reporter made it possible to isolate pure populations of Pax3 positive myogenic progenitor cells both in the embryo and in postnatal muscles where these cells assume a satellite cell position on muscle fibres. We have now carried out a transcriptome analysis of these quiescent cells in normal adult muscle, compared with activated satellite cells present in growing or damaged muscle. Previous analyses had been confined to comparisons with cultured cells; this in vivo analysis reveals important properties of the quiescent satellite cell and its niche on the muscle fibre as well as how it responds to activation.
We had previously described an unexpected transcription of the myogenic determination gene, Myf5, in specific regions of the central nervous system. We have now shown that this is due to a recent genome re-arrangement that introduces a regulatory sequence activated by Oct6 in the brain. The Myf5 protein is not present and we show that this is due to microRNA (miR) regulation; this provides a striking example of how the consequences of inappropriate transcription are avoided by this mechanism. We also show that Pax3 mRNA is specifically targeted by miR27 at the onset of muscle differentiation, in the embryo and the adult, thus providing a rapid and efficient way of down-regulating Pax3 which otherwise interferes with this process.
Research on cardiogenesis centers on our demonstration that two cell lineages contribute to the myocardium, and that there is a second heart field, characterized by a distinct gene regulatory network.
In the second heart field (SHF), FGF signaling plays an important role in the contribution of progenitor cells to the arterial pole of the heart. By manipulating a Sprouty transgene, we now show that FGF intervenes in an autocrine loop, such that the main recipient of the signal is the cardiac mesoderm itself; contrary to expectation, cardiac neural crest is not a primary target of FGF signaling. Analysis of Fgf8/Fgf10 conditional double mutants reveals a role for these ligands in pharyngeal arch artery formation as well as in the second heart field. Isolation of a cardiac enhancer in an intron of the Fgf10 gene, demonstrates that Tbx1 is required for expression in cardiac progenitors. Nkx2.5/Islet1 potentially modulate this genetic regulatory network. The transcriptional repressor Prdm1 also plays an important role in the second heart field.
Lineage studies on myocardial cells now focus on the venous pole of the heart where a retrospective clonal analysis is in progress. We have also initiated a prospective lineage analysis, with successful single cell injection of fluorescent markers in the mouse epiblast, to further characterize the segregation and cell fates of the first and second myocardial lineages.
Keywords: Myogenesis, Pax3 and Pax7, Myf5, cardiogenesis, second heart field
Montarras, D., Morgan, J., Collins, C., Relaix, F., Cumano, A., Partridge, T., and Buckingham, M. (2005) Direct isolation of muscle satellite cells demonstrates their major role in skeletal muscle self renewal. Science, 309: 2064-2067
Relaix, F., Rocancourt, D., Mansouri, A., & Buckingham, M.A. (2005). A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature, 435, 948-953.
Bajard, L., Relaix, F., Lagha, M., Rocancourt, D., Daubas, P., and Buckingham, M.E. (2006). A novel genetic hierarchy functions during hypaxial myogenesis : Pax3 directly activates Myf5 in muscle progenitor cells in the limb. Genes & Dev., 20, 2450-2464.
Prall, O.W.J., Menon, M.K., Solloway, M.J., Watanabe, Y., Zaffran, S., Bajolle, F., Biben, C., McBride, J.J., Robertson, B.R., Chaulet, H., Stennard, F.A., Wise, N., Schaft, D., Wolstein, O., Furtado, M.B., Shiratori, H., Chien, K.R., Hamada, H., Black, B.L., Saga, Y., Robertson, E.J., Buckingham, M.E., & Harvey, R.P. (2007). A Nkx2-5/Bmp2/Smad1 negative feedback loop archestrates cardiac progenitor cell specification and proliferation in the second heart field. Cell, 128, 947-959.
Lagha, M., Kormish, J.D., Rocancourt, D., Manceau, M., Epstein, J.A., Zaret, K.S., Relaix, F., & Buckingham, M.E. (2008). Pax3 regulation of FGF signaling affects the progression of embryonic progenitor cells into the myogenic program. Genes & Dev., 22, 1828-1837
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Activity Reports 2009 - Institut Pasteur
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