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  Director : Margaret BUCKINGHAM (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.

The regulation of the Myf5 gene has been analysed in transgenic mice, with YACs carrying large fragments of genomic DNA which include Myf5 targeted with an nlacZ reporter. With this approach we have identified different regions, extending to -96 kb upstream of Myf5, which control the spatiotemporal expression of this myogenic determination gene. Several enhancers are involved in its regulation, including one at -6 kb which is essential for early transcription of Myf5 in the epaxial dermomyotome of somites, before skeletal muscle begins to form. Another enhancer at -58/-48 kb is composed of several regulatory elements which we have shown target specific sites of Myf5 transcription in the somites, hypoglossal cord and limbs as well as in the nervous system, where the Myf5 protein is not detectable. The deletion of this enhancer in the context of the locus shows that it is essential. The analysis reveals the multiplicity of sequences which act together to orchestrate muscle formation. In the somite, for example, at least six sequences target distinct populations of cells which contribute to different phases of myogenesis. In this context, we are particularly interested in cells which participate in the formation of the intercalated region which lies at the border between the epaxial and hypaxial somite and which is characterized by the expression of En1. Another approach to understanding the regulation of Myf5, by an enhancer trap in cultured muscle cells, led to the isolation of an enhancer at -17 kb from Myf5, which has interesting properties. Just 5' to Myf5 there is a second gene encoding another myogenic factor, Mrf4, which has a different expression pattern. The sequence at -17 kb behaves differently with the two promoters. We have targeted Mrf4 with the alkaline phosphatase reporter which allows us to study the relation between the promoter of Mrf4 compared to that of Myf5 (targeted with nlacZ) and the other regulatory sequences of the locus. Deletion of the sequence at -17 kb suggests that it plays a key role in the organisation of the locus, favouring, for example, a configuration which permits the enhancer at -58/-48 kb to interact with the Myf5 promoter.

The characterisation of elements which target particular sites of myogenesis makes it possible to work backwards to the factors and signalling systems responsible for their activation and also to employ them to manipulate in vivo the network of regulatory molecules which specifies muscle identity in a cell population.

The targeting of Myf5 with an nlacZ reporter, by homologous recombination, provided important information about the essential role of this myogenic determination factor in the embryo. The Myf5-nlacZ allele is also expressed in satellite cells, required for the regeneration of adult muscle. Myf5 and MyoD are implicated in this process, but the Pax genes, and particularly Pax7, the orthologue of Pax3, are critical and cannot be complemented by Myf5. Targetting the Pax3 gene with an nlacZ reporter permitted us to show that Pax3 is also expressed in the satellite cellls of certain muscles. In the Pax7 -/- mutant there is a low level of regeneration and this is due to Pax3, expressed in the remaining satellite cells. We are examining the role of Pax3 in the proliferation and differentiation of these cells by manipulating Pax3, Pax7 and dominant negative or constitutively active forms, in adult muscle cultures. We are also interested in the existence of adult stem cells, and are looking at the myogenic/haematopoietic potential of bone marrow, as well as muscle, and at the possible role of Pax3 in directing the myogenic potential of mesangioblasts, present in the walls of blood vessels.

In the embryo, Pax3 plays a major role compared to Pax7, in skeletal muscle formation . By introducing the coding sequence of Pax7 into an allele of Pax3, also marked with the nlacZ reporter, we have examined the behaviour of cells which express Pax7 instead of Pax3. We find that Pax7 can only partially fulfil the functions of Pax3 The results are particularly interesting in the limbs, and point to the differences between fore- and hindlimb and between the formation of proximal and distal muscle masses.

Pax3 itself has little activity as a transcription factor and it is not clear whether it acts as a repressor or an activator in the embryo. A human chromosomal translocation leads to a fusion protein PAX3-FKHR which gives rise to a class of rhabdomyosarcomas. This protein, which keeps the DNA binding domain of PAX3, with the transactivation domain of FKHR, is a powerful transcriptional activator. We have targeted one allele of Pax3 with a PAX3-FKHR-IRESnlacZ sequence, and analysis of the resultant mouse shows that the fusion protein functions like Pax3 and can save the Pax3-/- mutant phenotype. Pax3 acts therefore as a transcriptional activator in the embryo, and we have isolated several candidate co-activators in this context. The protein PAX3-FKHR over-activates Pax3 targets such as c-met and the analysis of the behaviour of the cells which express this allele provides new insights into the function of Pax3 during myoenesis.

Not many targets of Pax3 are known and we are interested in Pax3-Six1/4-MyoD as a potential myogenic regulatory cascade. This is being examined by introducing a dominant negative form of Six into an allele of Pax3.

CardiogenesisRobert 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.

One transgenic line, in particular, has revealed the unexpected contribution of anterior mesodermal cells to the myocardium of the arterial pole of the heart. This phenomenon is confirmed by DiI labelling experiments in cultured embryos. 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 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, confirm that the primitive ventricle has a mainly "left" indentity.

Expression of the transgene in the anterior heart field is due to the integration site. It probably results from an "enhancer trap" type effect with 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, which will then be used to manipulate the expression of Fgf10 and other molecules of interest in the anterior heart field. The role of Fgf10 in this context is studied by analysing Fgf10 -/- embryos and by explant expriments.

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, visualised as b-galactosidase positive, provides information about the growth and behaviour of cells which form the myocardium. We distinguish a phase of dispersive growth, followed by a coherent phase, where clones are characterized by the particular orientation of cells to each other. In general cells within the same clone are distributed along the anterior/posterior axis of the heart. The distribution of labelled cells between different regions of the myocardium gives new insight into cardiac morphogenesis. In the context of the anterior heart field, for example, we observe a clonal separation between the right and left ventricle which is consistent with the different cellular origin proposed for these two regions of the heart.

Photo 1: The gene Pax3 targeted with an nlacZ reporter reveals defects in myogenesis and in the nervous system in mutant embryos A. An 11.5 day heterozygote embryo . B. An 11.5 day homozygote embryo. Red arrows show dorsal neural tube defects; green arrows show peripheral nervous system defects; black arrows show loss of migratory muscle precursors; white arrows show somitic defects.

Photo 2 :Cell lineage studies in the myocardium The labelling of cells derived from the same precursor (in blue) shown here for an embryonic mouse heart (OD: right atrium; OG: left atrium; VD: right ventricle; VG: left ventricle) gives new insight into the cellular mechanisms which underlie cardiac morphogenesis.

Keywords: Myogenesis, Cardiogenesis



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  personnel

  Office staff Researchers Scientific trainees Other personnel
  TAISNE Myriam (Institut Pasteur) - mtaisn@pasteur.fr Philippe DAUBAS (CNRS) - pdaubas@pasteur.fr

Robert KELLY (INSERM) - rkelly@pasteur.fr

Frédéric RELAIX (INSERM) - frelaix@pasteur.fr

Stéphane ZAFFRAN (CNRS) - zaffrans@pasteur.fr

Didier MONTARRAS (Institut Pasteur) - dmontarr@pasteur.fr

Ted CHANG - chang@pasteur.fr

Lola BAJARD - lbajard@pasteur.fr

Ralf SPOERLE - spoerle@pasteur.fr

Sigolène MEILHAC - meilhac@pasteur.fr

Catherine BODIN (Institut Pasteur) - cbodin@pasteur.fr

Dominique MICHEL - dmichel@pasteur.fr

Emmanuel PECNARD (CNRS) - pecnard@pasteur.fr

Jacqueline PERREAU (CNRS) - jperreau@pasteur.fr

Didier ROCANCOURT (Institut Pasteur) - drocanco@pasteur.fr


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