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

Skeletal myogenesis

Skeletal muscle development depends on a population of progenitor cells that express Pax3 and Pax7. In the absence of these factors, there is a major muscle deficit. In the embryo, Pax3 is dominant and we have carried out screens to identify Pax3 targets in order to understand the key role of this factor in myogenesis. These screens are based on the separation of cells by flow cytometry from mice with a Pax3^GFP allele, analysed on gain (Pax3-FKHR) or loss (Pax3-Engrailed) of function genetic backgrounds and show how Pax3 genetically regulates signaling pathways and notably inhibitors of signaling pathways that affect myogenesis. This has been investigated in more detail for Sprouty1 and Fgfr4 which we have shown to be a direct Pax3 target. Pax3 also controls genes encoding transcriptional regulators. We had demonstrated that the myogenic determination gene, Myf5, is targeted by Pax3, acting through a specific enhancer element. Pax3 activation of Dmrt2 also affects Myf5 via another early regulatory element acting at the onset of myogenesis in the embryo. Myogenic progenitor cells derive from the somite, which also gives rise to other mesodermal derivatives. Pax3 and Foxc2 are co-expressed in these multipotent cells, and we demonstrate reciprocal inhibition between these two genes. When the equilibrium between the transcription factors Pax3 and Foxc2 is perturbed, the choice of cell fate is affected. This is illustrated by interference with PKC mediated signaling, required for Pax3 activity which promotes the Foxc2-dependent vascular fate, at the expense of the Pax3-dependent myogenic cell fate. Foxc1 as well as Foxc2 is expressed in the somite and we are now analysing Foxc1/c2 double conditional mutants in the context of alternative cell fates.

Rapid down-regulation of Pax3 accompanies skeletal muscle differentiation. We had shown that this depends on microRNA27, which, when manipulated in the embryo or in satellite cells, which are the progenitors of post-natal skeletal muscle, affects the onset of myogenesis. We had also shown that microRNA31 targets the 3'UTR of Myf5 mRNA, preventing the inappropriate presence of this myogenic determination factor at sites where Myf5 is transcribed in the central nervous system. We have now found that this microRNA is transcribed in satellite cells and are investigating its potential role in modulating postnatal myogenesis. A transcriptome analyses of quiescent and in vivo activated satellite cells led us to show the importance of co-repressors, such as Dach1, in preventing premature myogenesis, of inhibitors of oxidative and genotoxic damage, and of metalloproteinase inhibitors, in the maintenance of the quiescent state. When the metalloproteinase activity is perturbed in skeletal muscle in vivo, activation of satellite cells and regeneration is compromised. The transcriptome analysis also revealed the presence of sequences for other transcription factors such as members of the Sox family or Pitx2/3, now under investigation.

Cardiogenesis

Research on cardiogenesis, in the mouse embryo, centers on our demonstration that two cell lineages contribute to the myocardium, and that there is a second heart field (SHF), characterized by a distinct gene regulatory network.

FGF signaling in the SHF plays an important role. Analysis of Fgf8/Fgf10 conditional double mutants shows the sensitivity of cardiogenesis to Fgf dosage, and reveals the role of Fgf10 as well as Fgf8 at the arterial pole of the heart, in pharyngeal arch artery formation as well as in the formation of the outflow tract. The transcriptional regulator, Prdm1, is also required for these vital processes, as shown by conditional mutant analysis.

Lineage studies, using retrospective clonal analysis, demonstrate that myocardium at the arterial pole of the heart and skeletal muscles in the head arise from a common progenitor cell. Sub-lineages distinguish categories of head muscles which segregate with pulmonary trunk or aortic derivatives of the outfow tract, or with right ventricular myocardium. A retrospective clonal analysis, extended to non-myocardial derivatives, that focusses on the venous pole is on-going. We have also initiated a prospective lineage analysis, based on single cell labeling in the mouse epiblast.

A study of cell behaviour during chamber morphogenesis is underway. This is based on our previous observations that oriented cell growth underlies heart morphogenesis. We now have evidence for the polarisation of myocardial cells and are investigating the role of the Fat4 planar cell polarity pathway. This study includes quantitative image analysis.

Keywords: Myogenesis, Satellite cells, Pax3 and Pax7, Foxc2, microRNAs, Cardiogenesis, Second heart field, Muscle cell lineages, FGFs

Lagha, M., Brunelli, S., Messina, G., Kume, T., Relaix, F., & Buckingham, M.E. (2009). Pax3/7:Foxc2 reciprocal repression in the somite modulates multipotent stem cell fates. Dev. Cell, 17, 892-899.

Lagha, M., Sato, T., Regnault, B., Cumano, A., Zuniga, A., Licht, J., Relaix, F., & Buckingham, M. (2010). Transcriptome analyses based on genetic screens for Pax3 myogenic targets in the mouse embryo. BMC Genomics, 11, 696.

Sato, T., Rocancourt, D., Marques, L., Thorsteindottir, S., & Buckingham, M. (2010). A Pax3/Dmrt2/Myf5 regulatory cascade functions at the onset of myogenesis. PloS Genetics, 6(4):e1000897.

Watanabe, Y., Miyagawa-Tomita, S., Vincent, S.D., Kelly, R.G., Moon, A.M., & Buckingham, M.E. (2010). Role of mesodermal FGF8 and FGF10 overlaps in the development of the arterial pole of the heart and pharyngeal arch arteries. Circ. Res., 106, 495-503.

Lescroart, F., Meilhac, S.M., Le Garrec, J.F., Nicolas, J.-F., Kelly, R.G., and Buckingham, M. (2010). Clonal analsys reveals common lineage relationships between head muscles and second heart field derivatives in the mouse embryo. Development, 137, 3269-3279.