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 (1). 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 Pax3GFP allele, analysed on gain (Pax3-FKHR) or loss (Pax3-Engrailed) of function genetic backgrounds (2). We had demonstrated that the myogenic determination gene, Myf5, is targeted by Pax3, acting through a specific regulatory element (3). Pax3 activation of Dmrt2 also affects Myf5, via another early enhancer (4). We continue to dissect Myf5 gene regulation, to identify which sequences are controlled by upstream factors such as Pax and Six. The critical balance between progenitor cell self-renewal and entry into the myogenic programme is orchestrated by Pax3 regulation of FGF signaling acting through genes for the inhibitor Sprouty1 and the receptor Fgfr4 (5). Myogenic progenitor cells derive from the somites, which also give rise to other mesodermal derivatives. Pax3 and Foxc2 are co-expressed in these multipotent cells and we demonstrate reciprocal inhibition between the 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 (6) [Figure 1]. We are now analysing conditional double mutants for Foxc2 and Foxc1, also expressed in the somite (collaboration with T. Kume). Other somite derivatives, such as brown fat, are under investigation, as well as the effects of Notch signaling on cell fate choices and myogenesis.

 


Figure 1: Cell fate decisions in multipotent cells of the embryonic somite.

Genetic analyses have demonstrated reciprocal repression between Pax3 and Foxc2 in multipotent cells of the somite as shown in the left hand panel. Modifying this equilibrium affects vascular versus myogenic cell fates as illustrated in the right hand panels. Signaling from the dorsal ectoderm which depends upon PKC, enhances Pax3 in the somite. In the presence of a PKC inhibitor (BIS), Pax3 and Pax7 mRNA levels are reduced, with a corresponding increase in Foxc2 miRNA, in somite explants. Skeletal muscle derivatives, marked by myosin heavy chain (MHC-green) are reduced with an increase in smooth muscle, marked by the labeling with an antibody to smooth muscle actin (a-Sma-red) in the absence of MHC.
  
 
In post-natal muscle, Pax7/(Pax3) positive satellite cells are responsible for muscle growth and regeneration (7) [Figure 2]. In the adult, these cells are normally quiescent, in their niche on the muscle fibre. A transcriptome analysis comparing quiescent versus in vivo activated satellite cells purified from Pax3GFP/+ mice has provided insights into the quiescent cell state and how this is modified on activation (8) [Figure 3]. Unlike muscle progenitor cells in the embryo, most quiescent satellite cells transcribe the Myf5 gene. The presence of this myogenic determination factor would lead to myogenic differentiation. We have shown that microRNA31 interacts with the 3' UTR of Myf5 mRNA to prevent inappropriate expression of the factor in the central nervous system at sites where the gene is transcribed (9). We are now investigating the role of miR31 in satellite cells, where it is also expressed. Muscle differentiation requires the rapid down-regulation of Pax factors and we have shown that this is effected by miR27 acting on Pax3 myogenic mRNA (10). On-going analysis of myogenic sequences targeted by miR27 reveals a regulatory network which includes Pax3 targets. It has recently been shown that, unexpectedly, Pax7 is not essential for adult skeletal muscle regeneration. We are currently examining conditional Pax3 mutants (collaboration with S. Conway) to clarify the role of Pax3 during post-natal myogenesis. Our transcriptome analysis shows that Pitx2 and Pitx3 are expressed in satellite cells. We are investigating conditional Pitx2/3 mutants (collaboration with J. Drouin) to determine the role of these transcription factors in muscle regeneration and to see whether they can substitute for Pax7/3 (collaboration with C.-M. Fan).

 
 
 
 
 
 
Figure 2: Visualisation and purification of muscle satellite cells.

 
A fluorescent satellite cell (GFP-green) lies within the basal lamina (marked by laminin-red) of adult muscle fibres of a Pax3GFP/+ mouse.

 

 
 
 

Figure 3: Characteristics of quiescent satellite cells in adult skeletal muscle.

The top panel shows a transcriptome analysis with high levels of transcript in red and low levels in blue in adult (ad-97% quiescent), adult regenerating (Ad.mdx-30.8% activated) and 1 week post-natal (1 wk - 81% activated) satellite cells, compared to the activated population of these cells in culture (Ad.cult). Quiescent cells express high levels of genes for Metalloproteinase (MMP) inhibitors, whereas their enzymes are high on activated cells. The lower panels illustrate how muscle regeneration is reduced in the presence of the MMP inhibitor (AM 409) which we propose reduces the liberation of satellite cells from their niche on the fibre and hence their activation.

 
 
 

Cardiogenesis

 
Research on cardiogenesis centers on our demonstration, in the mouse embryo, that two cell lineages (11) contribute to the myocardium, and that there is a second heart field (SHF), characterized by a distinct gene regulatory network (12).
Our analysis of regulation in the SHF has focused on FGF signalling which is essential for correct formation of the arterial pole of the heart, both of the outflow tract and pharyngeal arteries (13) [Figure 4]. The transcriptional regulator, Prdm1, is also required for these vital processes, as shown by conditional mutant analysis. On-going characterization of a SHF enhancer in the Fgf10 gene, provides insight into the control of expression in SHF progenitor cells versus repression in myocardial cells of the heart.
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 [Figure 5]. Sub-lineages distinguish categories of head muscles which segregate with pulmonary trunk or aortic derivatives of the outfow tract, or with right ventricular myocardium (14). 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.


 
Figure 4: The role of FGF signaling within the second heart field, in the formation of the arterial pole of the heart.

The left hand panel shows how defects in the embryonic outflow tract of the mouse heart (E10.5) become increasingly severe as Fgf gene dosage is reduced. [Fgf8flox is a conditional Fgf8 allele; mutation in cardiac progenitors of the second heart field is activated by crossing with a Mesp1Cre/+ line]. The right hand panels illustrate later defects (E16.5) at the arterial pole. Fgf8flox/+ serves as a control.

 
 
 
 

 
  Figure 5: Cell lineage relationships between head muscles and the arterial pole of the heart.

Cell polarity and dynamics in heart morphogenesis

 
With its precise 3D geometry, which is essential to orchestrate the circulation of the blood, the heart provides a striking example of morphogenesis. A study of cell behaviour and cell polarity underlying cardiac chamber expansion is underway. This is based on our previous observations on clones of myocardial cells, that showed that growth of the myocardium is oriented (15) [Figure 6]. Our aim is to understand how cell polarity is set up and how it regulates cell behaviour and ultimately the distinct shapes of cardiac chambers. We adopt interdisciplinary approaches, to map the polarity of myocardial cells in 3D and to model the formation of the embryonic heart.



 
Figure 6: Oriented growth of clones of myocardial cells
 
Example of a clone of
b-galactosidase positive cells in the ventricles of an embryonic heart from the a-cardiac actinnlaacZ1.1/+ mouse line, which shows the oriented growth of the myocardium. 
 
 
 
We now have evidence for the polarisation of myocardial cells and are investigating the role of planar cell polarity pathways (collaboration with H. McNeill) and primary cilia (collaboration with G. Pazour). We have developed tools for quantitative image analyses to extract polarity information (collaboration with J-C. Olivo-Marin) (16) as well as statistical methods to understand how cell polarity is coordinated at the level of the tissue.

Ongoing modelling of the morphogenesis of the embryonic heart provides insight into the respective contributions of different types of cell behaviour and of external mechanical constraints to the final shape of the organ (collaboration with E. Coen and A. Bangham) [Figure 7].

 

 

 

Figure 7: Computer simulation of heart morphogenesis. This simulation, from the straight heart tube to the looped heart tube, is based on a Finite Element Analysis model.

 

Selected publications from the Buckingham lab (cited in the text)

 

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

(2)     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.

(3)     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.

(4)     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.

(5)     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.

(6)     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.

(7)     Montarras, D., Morgan, J., Collins, C., Relaix, F., Zaffran, S., Cumano, A., Partridge, T., and Buckingham, M. (2005) Direct isolation of satellite cells for skeletal muscle regeneration. Science, 309: 2064-2067.

(8)     Pallafacchina G., François, S., Regnault, B., Czarny, B., Dive, V., Cumano, A., Montarras, D., & Buckingham, M. (2010). An adult
tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells. Stem Cell Res., 4,
77-91.

  (9)    Daubas, P., Crist, C.G., Bajard, L., Relaix, F., Pecnard, E., Rocancourt, D., & Buckingham, M. (2009). The regulatory mechanisms that
underlie inappropriate transcription of the myogenic determination gene Myf5 in the central nervous system. Dev. Biol., 327, 71-82.

(10)    Crist, C.G., Rocancourt, D., Montarras, D., & Buckingham, M. (2009). Muscle stem cell behaviour is modified by microRNA-27 regulation of Pax3 expression. Proc. Natl. Acad. Sci. USA, 106, 13383-13387.

(11)  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.

(12)  Buckingham, M., Meilhac, S., and Zaffran, S. (2005). Building the mammalian heart from two sources of myocardial cells. Nat. Rev. Genet., 6, 826-835.

(13)    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.

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

(15)    Meilhac, S.M., Esner, M., & Buckigham, M.E. (2004). Oriented clonal cell growth in the developing mouse myocardium underlies cardiac morphogenesis. J. Cell Biol., 164, 97-109.

(16)    Pop, S., Dufour, A., Le Garrec, J-F., Ragni, C., Buckingham, M., Meilhac, S. and Olivo-Marin, J-C. (2011). A fast and automated framework for extraction of nuclei from cluttered 3D images in fluorescence microscopy. Proceedings of IEEE International Symposium on Biomedical Imaging: from Nano to Macro - ISBI2011, p2113-2116, Chicago, 29 March -- 2 April 2011