Unit: Molecular Genetics of Development - URA CNRS 2578
Director: Margaret BUCKINGHAM
Our research focusses on the study of myogenesis, with the aim of understanding how muscle cells are specified in the embryo and how this leads to the formation of different skeletal muscles. These studies extend to muscle progenitor cells in the adult and the potential contribution of stem cells to skeletal muscle regeneration. We are also interested in cardiogenesis, with the analysis of the origin and behaviour of different populations of myocardial precursor cells and how they contribute to heart morphogenesis. The animal model is the mouse, using experimental approaches based on gene manipulation.
The formation of skeletal muscle
Myf5 and Pax3 are upstream genes in the genetic hierarchy that determines the entry of a cell into the myogenic differentiation programme. Myf5 is a member of the family of b-HLH myogenic regulatory factors (Myf5, Mrf4, MyoD and Myogenin), while Pax3 belongs to the Pax family of homeo-domain transcription factors, members of which are implicated in the appearance of specialized cell types, such as B lymphocytes, during development.
Myf5 gene regulation
[Lola Bajard, Ted Chang, Philippe Daubas, Didier Rocancourt, Ralf Spörle [Collaboration with P. Rigby (London)].
We are studying Myf5 regulation, with the aim of understanding how this myogenic regulatory gene is activated in the embryo. By a transgenic approach, using YACs carrying large fragments of DNA, we have identified different regions, up to -96 kb upstream of Myf5, which control the spatio-temporal expression of the gene. In particular a sequence located between -58/-48 kb is composed of elements which target specific sites of Myf5 transcription in somites and limbs, as well as in regions of the C.N.S. where the protein is not detectable. The deletion of this enhancer, in the context of the locus, shows its essential role. This analysis reveals the multiplicity of regulatory sequences which orchestrate the formation of skeletal muscle from different myogenic precursors. We are now looking for the activators of such sequences.
(Mounia Lagha, Frédéric Relaix, Didier Rocancourt [Collaboration with B. Schafer (Zurich), C. Ponzetto (Turin) et Ahmed Mansouri (Göttingen)].
In splotch mutants, the Pax3 gene is mutated and muscle masses are absent, including those in the limbs. Targeting Pax3 with an nlacZ reporter gene has allowed us to follow the cells which express the gene in normal and mutant embryos. Pax3 itself shows little transcriptional activity and it is not clear whether it acts as a transcriptional activator or repressor in vivo. In man, a chromosomal translocation leads to a fusion protein, PAX3-FKHR, implicated in the formation of rhabdomyosarcomas. This protein which comprises the DNA binding domain of PAX3 and the transactivation domain of FKHR, is a strong transcriptional activator. We have targeted an allele of Pax3 with the sequence PAX3-FKHR-IRESnlacZ and shown that PAX3-FKHR functions like Pax3, in that it rescues the Pax3-/- mutant phenotype both in skeletal muscle and in neural crest where the gene is also expressed . Pax3 therefore acts as a transcriptional activator in the embryo, as also indicated by the expression of a transgene regulated by Pax3 binding sites. Embryos which express PAX3-FKHR overactivate MyoD and c-met, confirming that they are Pax3 targets. They also demonstrate the anti-apoptotic effect of Pax3 ; loss of cells complicated target identification in the Pax3 loss of function mutant. The over-expression of c-met provokes an ectopic delamination of muscle precursors from thoracic somites, with activation of the Met receptor in the absence of the ligand HGF - a phenomenon of interest embryologically and also in the context of tumorigenesis. We are currently looking for other Pax3 targets, using the gain and loss of function mutants.
(Didier Montarras, Frédéric Relaix, Didier Rocancourt) [Collaboration with A. Cumano (IP) and T. Partridge, London)].
Satellite cells, located under the basal lamina of muscle fibres, are the precursors of adult muscle. Pax7, the orthologue of Pax3, has been shown to play an important role in these cells. Our Pax3-nlacZ reporter mice show that Pax3 is also expressed in the satellite cells of certain muscles. In the absence of Pax7, satellite cells are present and capable of forming skeletal muscle, but in greatly reduced numbers. The construction of a Pax3-GFP mouse now facilitates the isolation of these cells by FACS sorting and the study of the role of Pax3 in the adult.
mesoangioblasts and the dorsal aorta
(Milan Esner, Sigolène Meilhac, Didier Montarras) [Collaboration with G. Cossu (Milan), A. Cumano (IP) et J.-F. Nicolas (IP)].
Several sources of stem cells which can contribute to skeletal muscle have been proposed, among which mesoangioblasts, derived from the walls of blood vessels are particularly interesting. We are studying these cells, which express Pax3. They first appear in the dorsal aorta in the embryo and we are carrying out a lineage study, based on the use of a genetic marker which labels both cells of the aorta and muscle cells in the somite, to see if they are derived from a common precursor.
(Fany Bajolle, Milan Esner, Sigolène Meilhac, Emmanuel Pecnard, Stéphane Zaffran) [principal collaborations : N. Brown (London), R. Kelly (New York) et J.-F. Nicolas (IP)]
Cardiogenesis depends on regulatory genes which give often unexpected mutant phenotypes affecting specific compartments of the heart. Another important parameter in this process is cellular and concerns both the origin and the behaviour of cells which will contribute to different parts of the heart. In order to examine this aspect we have developped cardiac actin-nlaacZ mice in which a rare recombination event in a cell leads to an nlacZ allele with production of β-galactosidase. This permits a retrospective clonal analysis of myocardial cells, all of which express cardiac actin. We have first shown that labeled cells in a clone derived from an early recombination event are dispersed on the anterior/posterior axis of the cardiac tube. This result is not compatible with a segmental model in which the organisation of cardiac precursor cells in the early mesoderm already reflects the ordering of cardiac compartments. In contrast, we show that later labeled cells are present in clusters and thus that a coherent phase follows the dispersive phase of cell growth . Analysis of coherent growth shows that the form of each cardiac compartment is prefigured by oriented cell growth which distinguishes cells in the corresponding part of the cardiac tube.
This clonal analysis also gives important insight into the origin of myocardial cells. We distinguish two lineages which contribute to all parts of the heart, with the exception, in the case of the first, of the outflow region, and in the case of the second, the future left ventricle. These results are consistent with our previous observation of an unexpected contribution of pharyngeal mesoderm ("anterior heart field") to outflow tract myocardium. Recently, using explants of pharyngeal mesoderm or of the cardiac tube from transgenic mice which mark different parts of the heart, we have demonstrated that most of the primitive cardiac tube has a left ventricular identity ; pharyngeal mesoderm contributing myocardial cells with characteristics of the right ventricle as well as outflow tract.
Ongoing work includes a fine analysis of the morphogenesis of the outflow tract, as well as the formation of the atria and their acquisition of left/right identity. We are also interested in mutants which affect heart morphogenesis, in the context of different myocardial cell precursors.
Figure 1 PAX3-FKHR rescues the Pax3 mutant (Sp) phenotype.
A,C. Normal distribution of β-galactosidase positive cells.
B. Loss of limb muscles (black arrowhead) and somite abnormalities (red arrowhead) in the absence of Pax3.
D. Restoration of the phenotype shown in B, by PAX3-FKHR.
Figure 2 Two phases of clonal cell growth in the cardiac tube, visualised at E8.5.
A. A single cluster of β-galactosidase positive cells showing coherent growth.
B. Several clusters of β-galactosidase positive cells illustrating dispersive cell growth of a myocardial progenitor cell.
Keywords: Myogenesis, Pax3, Myf5, Cardiogenesis, Cell Lineage