Molecular Genetics of Development - CNRS URA2578  


  HEADProf. BUCKINGHAM Margaret / margab@pasteur.fr
  MEMBERSDr BAJARD Lola/BODIN Catherine/Dr CRIST Colin
Dr DAUBAS Philippe/Dr ESNER Milan/LAGHA Mounia
LESCROART Fabienne/MARCHISET Sophie/Dr MEILHAC Sigolène
Dr MONTARRAS Didier/Dr PALLAFACCHINA Giorgia/PECNARD Emmanuel/ ROCANCOURT Didier/Dr SATO Takahiko
TAISNE Myriam/Dr TOMITA Sachiko/Dr VINCENT Stéphane/Dr WATANABE Yusuke


  Annual Report

Our Unit works on myogenesis and cardiogenesis, using the tools of mouse molecular genetics to examine cell behaviour and gene function.

Skeletal myogenesis

We are focussing on the upstream signalling pathways and transcriptional regulators that control the entry of a cell into the myogenic programme, both during skeletal muscle formation in the embryo and regeneration in the adult.

We had established genetically that Pax3 and Pax7 play an essential role in ensuring the survival and the myogenic potential of muscle progenitor cells. In the embryo, myogenesis is initiated in the somites. Non-canonical Wnt signalling from the dorsal ectoderm, acting through protein kinase C (PKC) results in the activation of Pax3 in the hypaxial somite. This results in the transcription of the myogenic determination gene, MyoD, leading to myogenesis (collaboration with S. Brunelli, G. Cossu). In this context, the transcriptional activity of Pax3 probably depends on activation of co-factors. We are currently developing an in vivo approach for the isolation of Pax3 co-factors at different sites of myogenesis. Activation of the myogenic determination gene, Myf5, also depends on Pax3, which in this case acts directly on a regulatory element (-57.5 kb) required for transcription of Myf5 in the developing limb buds. We now show that Six 1/4, members of the Six homeodomain family of transcription factors, also control the activity of this element, consistent with their role, together with Pax3, as upstream regulators of myogenesis (collaboration with P. Maire).

A second myogenic regulatory gene, Mrf4, is also present in the Myf5 locus. We have characterized a control element (-17 kb) that differentially regulates these two genes. In adult skeletal muscle, with the Myf5 promoter, this element directs Myf5-like transcription in progenitor (satellite) cells, whereas with the Mrf4 promoter, expression is directed to the myonuclei of differentiated muscle fibres (collaboration with P. Zammit).

We continue to study the Mrf4-Myf5 locus as an entry point into the myogenic programme. However much of our current work is also directed to identifying other Pax3 targets that modulate myogenesis in the embryo, together with microarray analyses on progenitor cells isolated from post-natal skeletal muscles of Pax3GFP/+ mice.

Cardiogenesis

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), analysis of Nkx2.5-/- embryos, shows that this transcription factor, which is already expressed in cardiac progenitor cells, controls a negative feedback loop with Bmp2/Smad1, that regulates the expansion of this population and its contribution to the heart. Furthermore, in the absence of Nkx2.5, markers of the SHF continue to be expressed in differentiating myocardium (collaboration with R. Harvey).

The arterial pole of the heart derives from the anterior SHF. We show that subdomains of the outflow tract myocardium have distinct transcriptional programmes which are later restricted to myocardium at the base of the pulmonary artery or aorta, that derive from this region. In parallel, retrospective clonal analysis shows coherent clonal growth in the outflow tract, with limited intercalation, and subsequent clonal restriction to the base of one of the great arteries. Large clones indicate that cell intermingling is also restricted in the anterior SHF.

Current research on the formation of the outflow tract focusses on the role in the anterior SHF of FGF signalling, and of the transcriptional repressor Blimp1 (prdm1), also expressed during early skeletal myogenesis. A detailed analysis of the venous pole of the heart is on-going, as well as a prospective clonal analysis of myocardial progenitors in the early mouse embryo.

Keywords: Myogenesis, Pax3 and Pax7, Myf5, cardiogenesis, second heart field

Gmd.jpg

Clonally related, b-galactosidase positive (blue) cells are shown in superior (A) or inferior (B) aspects of the ouflow tract of the mouse heart at E10.5 and later around the base of the pulmonary trunk (C) or aorta (D) at E14.5, thus illustrating restricted mingling of these myocardial cells and their progenitors.



  Publications

Meilhac, S.M., Esner, M., Kelly, R.G., Nicolas, J-F., & Buckingham, M.E. (2004). The clonal origin of myocardial cells in different regions of the embryonic mouse heart. Dev. Cell, 6, 685-698.

Relaix, F., Rocancourt, D., Mansouri, A., & Buckingham, M.A. (2005). A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature, 435, 948-953.

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.

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.



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Activity Reports 2007 - Institut Pasteur
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