Unit: Molecular Genetics of Development
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.
Myf5 and Mrf4 genes, as well as Pax3 and its paralogue Pax7, act upstream in the genetic hierarchy that determines the entry of a cell into the myogenic differentiation programme. Myf5 and Mrf4 are members 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 play a key role in organogenesis and in the appearance of specialized cell types during development.
Regulation of the Mrf4-Myf5 locus
[Lola Bajard, Ted Chang, Philippe Daubas, Didier Rocancourt [Collaboration with P. Zammit (London)].
We are studying the regulation of the Mrf4-Myf5 locus, with the aim of understanding how these myogenic determination genes are activated in the embryo. By a transgenic approach, we have identified different regions, extending -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 central nervous system where the protein is not detectable. We have characterised an element that directs expression of Myf5 in the limb buds and have identified a candidate regulatory molecule.
Another regulatory element, situated at -17 kb from Myf5, directs transcription in the intercalated myotome or in ftal muscle, depending on the presence of the Mrf4 or Myf5 promoter. This promoter specific regulation, found in the embryo, is also striking in the adult, with expression in muscle fibres or in satellite cells, respectively.
Pax3 function, compared to Pax7
[Mounia Lagha, Didier Montarras, Frédéric Relaix, Didier Rocancourt (Collaboration with Ahmed Mansouri (Göttingen)].
In the absence of Pax3, several categories of muscles are missing including those of the limbs. Neural crest derivatives are also affected. The introduction of an nLacZ, and more recently a GFP, reporter into Pax3, has permitted us to follow the cells that express the gene. Pax7, the paralogue of Pax3 is expressed in a subpopulation of Pax3 positive cells in the embryo. In order to compare the functions of Pax7 in relation to Pax3 we have introduced the coding sequence of Pax7 into the Pax3 gene, accompanied by a reporter sequence (IRES-nLacZ). We observe that all the functions of Pax3 in neural crest and in muscle in the trunk, are replaced by Pax7. In contrast, skeletal muscle formation in the limb is affected, with problems of migration and replication of myogenic progenitor cells derived from the somites (Figure 1). From an evolutionary point of view it is interesting that in a cephalochordate, such as Amphioxus, there is only a single Pax3/7 gene which is expressed in neural crest like cells, in somites and axial muscles. On the basis of our observations, we suggest that Pax3 acquired specific functions required for limb development during tetrapod radiation.
In adult muscle, Pax7 is expressed in the progenitor cells, known as satellite cells. In the Pax7 mutant mouse, these cells are absent and it was proposed that Pax7 is necessary for their specification. We show that these cells are present after birth, but that they die progressively. Pax3 is also expressed, in a subpopulation of satellite cells. While Pax3, like Pax7, plays a key role in the activation of the myogenic programme in these cells, Pax7 has an additional anti-apoptotic role in postnatal skeletal muscle.
The regulation of the cardiac actin gene
[Marguerite Lemonnier, Sigolène Meilhac, Susanna Molinari, Frédéric Relaix (Collaboration with B. Schaeffer (Zurich)]
Cardiac actin is expressed in skeletal muscle and in certain smooth muscle cells in the embryo, as well as in cardiac muscle. We have identified regulatory sequences upstream of the gene, and in particular an enhancer which is activated by Mef2 and which specifically directs transcription in embryonic and adult myocardium. Another enhancer which directs expression only in skeletal muscle, depends on myogenic regulatory factors and on a complex formed with Mef2D, the histone transacetylase CBP/p300 and a protein, Emb, of the POU domain family of transcription factors. This complex, as well as the role of Emb isoforms in skeletal muscle, are of interest in the context of chromatin control of the cardiac actin locus.
The cardiac actin gene was targeted with an nLaacZ reporter, which after a rare event of intragenic recombination, can be converted into a functional nLacZ sequence. Mice that carry this targeted allele have proved to be a valuable tool for retrospective clonal analysis in different types of muscle, where the cardiac actin gene is expressed.
Stem cells-mesoangioblasts and the dorsal aorta
[Milan Esner, Sigolène Meilhac (Collaborations with Giulio Cossu (Milan), Ana Cumano (IP) and Jean-François Nicolas (IP)]
Several sources of skeletal muscle stem cells have been proposed, among which mesoangioblasts, derived from blood vessels, are particularly interesting. These cells express Pax3. In the embryo, they have been isolated from the dorsal aorta, which is also a source of hematopoietic stem cells. We are studying possible links between these two types of stem cells.
In the context of mesoangioblasts, we have carried out a lineage analysis between cells in the dorsal aorta and in the myotome, the first skeletal muscle to form, within the somites. Mice in which the cardiac actin gene has been targeted with nLaacZ have been employed to carry out a retrospective clonal analysis which demonstrates that cells in the dorsal aorta and in the myotome are derived from a common progenitor. Most of these cells, dispersed around the wall of the aorta, express smooth muscle markers, but rare endothelial cells are also present in the clones, providing proof for a common vascular progenitor.
[Fanny Bajolle, Milan Esner, Sigolène Meilhac, Emmanuel Pecnard, Stéphane Zaffran (Collaborations principally with Nigel Brown (London), Robert Kelly (New York) and Jean-François Nicolas (IP)]
We have shown, in the mouse, that there is a second heart field, situated in pharyngeal mesoderm and providing a source of myocardial cells that contribute to the arterial pole of the heart. This discovery was based on observations on transgenic mouse lines and on the tracing of DiI labelled cells. More recently, retrospective clonal analysis at E8.5 with the cardiac actinnLaacZ/+ mouse line, has led us to identify two myocardial cell lineages. Both contribute to all parts of the heart, with the exception, for the first, of the outflow tract, and, for the second, of the left ventricle. These lineage results indicate that the second heart field contributes more extensively to the myocardium, including the right ventricle and the venous pole. Another experimental approach has also shown a difference in the formation of the two ventricles. Explant cultures of pharyngeal mesoderm or of the cardiac tube, derived from transgenic mouse lines which mark different regions of the heart, show that most of the primitive heart tube has a left ventricular identity. The progenitor cells of the right ventricle as well as the outflow tract are present in pharyngeal mesoderm. These results were confirmed by DiI labelling experiments with cultured mouse embryos.
The retrospective clonal analysis also permitted us to characterise the behaviour of myocardial progenitor cells. After an initial period of dispersive growth, myocardial cells at E10.5 and already at E8.5 show coherent and orientated cell growth (Figure 2). This is one of the first examples of orientated cell growth in vertebrates, which we show prefigures the form of the different compartments of the mouse heart.
The analysis of clones in the outflow tract of the heart at different developmental stages, provides insight into a dorsal/ventral regionalisation which is also indicated by transgenic markers. These tools provide the demonstration of a rotation of the outflow tract myocardium during the maturation of this region of the heart, also shown by DiI labelling. This phenomenon has important consequences for the alignment of the great vessels and the interpretation of congenital malformations which frequently affect the outflow tract region of the heart.
Figure 1: Expression at E12.5 of β-galactosidase in a Pax3Pax7-IRESnLac/+ embryo, showing normal muscle development in the trunk (A) and forelimb (C) when one functional Pax3 allele is present. In contrast in a Pax3Pax7-IRESnLacZ/Sp embryo in which no Pax3 alleles are present and one Pax3 allele is replaced by Pax7, trunk musculature is normal (B), but forelimb muscles are missing (D).
Figure 2: Clusters of β-galactosidase positive cells in the right ventricle of an α-cardiac actinnLaacZ/+ mouse heart at E10.5 demonstrate oriented cell growth emanating from a central * point towards the expanding periphery of this ventricular chamber.
Keywords: Myogenesis, Pax3, Pax7, Myf5, Cardiogenesis, Dorsal Aorta, Cell Lineages