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  Director : BUCKINGHAM Margaret (margab@pasteur.fr)


  abstract

 

Our research centres 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 muscle masses, both skeletal and cardiac, during development. We are also interested in muscle progenitor cells in the adult and the potential contribution of stem cells to regeneration. The experimental model is the mouse, with genetic manipulation of regulatory genes.



  report

cale

The formation of skeletal muscle [Lola Bajard, Ted Chang, Philippe Daubas, Jacqueline Perreau, Frédéric Relaix, Didier Rocancourt, Ralf Spörle, Shahragim Tajbakhsh (independent group since 10/01) — collaborations with the laboratories of G. Cossu, P. Maire, A. Mansouri, T. Partridge, M. Polimeni, P. Rigby, , B. Schäfer]

In 1997 we showed that Myf5 and Pax3 intervene upstream of MyoD in the genetic hierarchy that regulates the onset of myogenesis. Our current research focusses on the regulation and function of these genes.

We have analysed Myf5 regulation with YAC transgenes which carry large fragments of DNA from around the locus. Several regions within 96 kbp upstream of the gene have been identified which are necessary for the spatio-temporal pattern of expression in the mouse. These include several enhancers one of which at —58/-48 kbp from Myf5 is composed of distinct sub-regions, directing reporter (nlacZ) gene expression in the myotome compartment of somites, in trunk and limb muscles and in those regions of the central nervous system where we had previously reported unexpected transcription of this myogenic regulatory gene. The earliest expression of Myf5 in somites depends on another enhancer, located at —6 kbp; deletion of this element in the context of the locus (200 kbp) specifically eliminates Myf5 transcription in the epaxial dermomyotome, but not in the myotome, which is the target of other myogenic regulators. A sequence at —17 kbp also acts as an enhancer, directing expression to a subdomain of the myotome which is of particular interest. Located between the epaxial and hypaxial parts of the somite the corresponding intercalated domain of the dermomyotome is characterized by the expression of markers such as Engrailed 1, implicated in boundary formation in the embryo. Recently we have identified other markers which permit a fine analysis of the dynamic nature of this domain during somite maturation in the embryo. Deletion of the —17 kbp enhancer gives an interesting result. A second gene, encoding another myogenic regulatory factor, Mrf4, is located at —6 kbp from Myf5 and probably shares some of the upstream regulatory elements, although its expression pattern is not the same. The introduction of a second reporter gene (alkaline phosphatase) in Mrf4 makes it possible to follow the relation between the promoters of Myf5 or Mrf4 and other regulatory elements/enhancers. Our preliminary results suggest that the element at —17 kbp plays a key role in the regulatory organisation of the locus.

Genetic manipulation of the Myf5 gene, with the introduction of an nlacZ reporter, revealed the role of this myogenic factor in the positioning of myogenic progenitor cells required for the formation of the myotome, as well as in the initiation of the regulatory cascade which leads to their differentiation into muscle. The first mutants that we and others obtained died at birth due to respiratory problems resulting from deficient rib formation. A second generation of mutants, with the selectable marker gene, neo, removed, do not have this rib phenotype and are viable. Our analysis of this phenomenon shows that it depends on the position as well as the nature of the insertion in the locus, probably due to a cis-acting effect on another gene directly or indirectly involved in rib formation. There is a correlation between perturbations in Mrf4 transcription and the rib phenotype suggesting that this factor, which plays a role in the differentiation of myotomal muscle, is a candidate. These observations underline the complex relationships between Mrf4 and Myf5 gene regulation.

The Myf5-nlacZ allele is also expressed in satellite cells which are associated with adult muscle fibres. The regeneration of damaged muscle takes place as a result of satellite cell activation. Pax7 and/or its orthologue Pax3 is also expressed in these cells. In 2000, M. Rudnicki and colleagues showed that satellite cells are absent in a Pax7-/- mutant, in limb muscles where Pax7 predominates. Contrary to what happens during myogenesis in the embryo, Myf5 does not complement the absence of Pax at the level of muscle cell specification. We are examining the role of Pax3 in the satellite cells of muscles such as the diaphragm where we have shown this Pax gene to be expressed. As in the case of Myf5 the manipulation of the locus, with the introduction of reporter genes, enables us to follow the cell populations which express Pax3 in adult mice and also in normal and mutant embryos.

Pax3 is expressed in presomitic mesoderm as well as in muscle progenitor cells. The targetting of other sequences to the Pax3 gene therefore allows us to manipulate early events leading to myogenesis, both at the level of the signallling systems which activate the myogenic determination factors and of other transcription factors implicated in the regulatory cascade which implements the formation of skeletal muscle.

The signalling pathways which control the initiation of myogenesis have already been investigated in the laboratory. The myogenic regulatory sequences of Myf5 are studied in this context. In order to examine a potential role of Notch signalling in myogenic cell specification we have targetted alleles of Pax3 and Myf5 with a sequence encoding a constitutively active, intracellular form of Notch. Perturbations in muscle formation and in neural crest derivatives, particularly evident in the case of the Pax3 allele, are under study.

Members of the Six family of homeoproteins are necessary for the activation of genes involved in muscle cell differentiation and probably also in the earlier events leading to MyoD activation by Pax3. We have targetted an allele of Pax3 with a sequence encoding a dominant negative form of Six4 in order to investigate a potential upstream role of these factors in myogenesis. Embryos with this allele are currently being analysed.

Cardiogenesis

Robert Kelly, Marguerite Lemonnier, Sigolène Meilhac, Stéphane Zaffran — collaborations with the laboratories of N. Brown et J.-F. Nicolas]

The regionalized expression of certain transgenes which are expressed in cardiac muscle, provided us with an interesting tool with which to follow different subdomains of the myocardium, both in vivo during the development of the heart, and in vitro. Thus in explants of the early cardiac tube we are able to identify a time frame when left/right atrial identity is still flexible whereas that of the ventricule is already acquired. We have constructed adenovirus vectors expressing different signalling molecules and factors which enable us to manipulate this acquisition of identity on the left/right and, potentially also, on the anterior/posterior axis.

In most cases regulatory sequences of the transgenes are responsible for their regionalized transcription in the heart. We are trying to define these sequences by transgenic experiments in vivo. We conclude, based on in vitro manipulation of cardiomyocytes, that expression of the transgene, integrated in the genome, becomes unalterable, probably due to chromatin organisation, at an early stage of cardiogenesis, perhaps at the time when myocardial cells acquire positional identity, as monitored in the explant experiments.

One transgenic line, in particular, has revealed the unexpected contribution of anterior mesodermal cells to the myocardium at the arterial pole of the heart. This phenomenon is confirmed by DiI labelling experiments in embryo culture. The origin of this part of the heart had remained obscure. It is now clear from our observations with mouse embryos, and from recent work from other laboratories on the chick, that there is a second source of cardiac precursor cells, initially situated medially to the cardiac crescent where the first cardiomyocytes differentiate, and then, following the movement of cell masses which accompany the development of this region, lying anteriorly to the cardiac tube which forms by fusion of the crescent and which grows by the addition of cells caudally. The cells coming from the anterior heart field, that we have identified, contribute to the anterior part of the cardiac tube to form the outflow tract and probably also at least part of the right ventricle. Explant experiments both with the line where expression of the transgene marks the progenitor cells of this region and with transgenic lines where other regions of the myocardium are marked, allow us to confirm and study the contribution of the anterior heart field.

In contrast to other transgenic lines with regionalized expression in the heart, expression of the transgene in the anterior heart field is due to the integration site. It probably results from an "enhancer trap" type effect from the Fgf10 gene which lies in the same genomic region and which has a similar pattern of transcription in the anterior heart field. We are currently trying to identify the regulatory elements in question. The long half-life of b-galactosidase provides a chase of transgene expression into the arterial pole myocardium. In the presence of an inhibitor of Fgf signalling in cultured embryos this process is perturbed and truncations of the outflow tract are observed, pointing to a role for Fgf10 in the migration/proliferation of the precursor cells for this part of the heart. Another Fgf, Fgf8, is also present in the anterior heart field and may explain why Fgf10-/- mutants do not have a pronounced cardiac phenotype; these mutant embryos are now being re-examined. Our transgenic line also has a phenotype, characterized as a hypomorph of Fgf10.

The question of the origin of the cell populations which colonise different parts of the developing heart is also being addressed by a retrospective clonal analysis. We have targetted the cardiac actin gene with an nlaacZ reporter sequence in which a rare combination event will lead to a functional nlacZ and consequent labelling with b-galactosidase of a cell expressing the cardiac actin allele. The analysis of clones in the embryonic heart visualised in this way already shows that the pool of founder cells is large. The results indicate clonal regionalisation between the right and left ventricles which may reflect the different histories of these two cardiac compartments. The comparison of clones in skeletal muscle, also analysed with this transgene, shows interesting differences. For example, an analysis during the perinatal period, occasionally shows almost complete labelling of a single muscle, suggesting an oligoclonal origin.



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  personnel

  Office staff Researchers Scientific trainees Other personnel
 

Myriam TAISNE (Institut Pasteur)

Philippe DAUBAS (CNRS)

Robert KELLY (INSERM)

Marguerite LEMONNIER (INSERM) – jusqu'en novembre 2001

Frédéric RELAIX (INSERM)

Shahragim TAJBAKHSH (Institut Pasteur) – jusqu'en septembre 2001, à partir de 10/01, responsable d'un groupe indépendant.

Ted CHANG (postdoc)

Lola BAJARD (étudiante en DEA) – depuis septembre 2001

Ralf SPÖRLE (postdoc)

Sigolène MEILHAC (étudiante en thèse)

Stéphane ZAFFRAN (postdoc)

Catherine BODIN (IP)

Dominique MICHEL (IP)

Jacqueline PERREAU (CNRS)

Didier ROCANCOURT (IP)


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