Unit: Morphogenesis Molecular Genetics - URA CNRS 2578
Director: ROBERT, Benoît
Our laboratory is studying the function of Msx homeobox genes, using mouse mutants that we have produced. These genes are involved in the transduction of molecular signals (BMPs, WNTs ) at major sites of embryonic induction, such as the apical region of the limb buds or the dorsal midline of the neural tube. Some data further suggest that their expression may characterise stem cells. Through the analysis of these genes, we are involved in the study of fundamental mechanisms of embryonic induction and of cell plasticity.
The laboratory of Molecular Genetics of Morphogenesis is involved in the study of properties and function of the Msx1 and Msx2 homeobox genes. These genes are prominently expressed at sites of induction between ectoderm and mesoderm. We have inactivated both Msx1 and Msx2 by inserting an nlacZ reporter gene in each locus, which permits to follow their expression during development and facilitates the analysis of mutant phenotypes. Msx1:Msx2 double mutants have led to a considerable extension of these analyses. These mutants confirm the implication of Msx genes in inductive signal transduction required for organogenesis in vertebrates, particularly for limb outgrowth and morphogenesis and the formation of the dorsal midline of the neural tube.
Role of Msx genes in limb morphogenesis (Yvan Lallemand, Vardina Bensoussan, Cécile Saint Cloment)
Homozygous Msx1:Msx2 double mutants exhibit severe limb defects. Anterior elements of the limb skeleton (radius, tibia) fail to form. In the autopod, the situation is more complex: anterior elements (such as the thumb) are often missing, but paradoxically, polydactyly is not rare. We have demonstrated that at early stages, Msx genes are required to specify dorso-ventral polarity in the limb bud anterior region. Dorsal or ventral identity depends on BMP2, 4 and 7 signalling, and Msx genes are required for BMP signal transduction. Due to lack of dorso-ventral polarity, the apical ectodermal ridge does not form anteriorly, and consequently, limb outgrowth and formation of anterior skeletal elements are impaired in Msx1: Msx2 double mutants. At later stages, outgrowth of the autopod and formation of anterior polydactyly is often observed, that may also be related to BMP signalling. Extra tissues nonetheless keep a posterior identity, and we have demonstrated that Msx genes are required for anterior specification. In the limb, antero-posterior polarisation depends mainly on Shh / Gli3 signalling. Our data unravel genetic interactions between these and Msx genes, demonstrating that Msx are critically involved in several signalling pathways.
Role of Msx genes in the formation of the dorsal midline of the neural tube (Yvan Lallemand, Matthieu Genestine, Cécile Saint Cloment)
Msx1 and Msx2 are also expressed in the dorsal midline of the neural tube (roof plate). We showed previously that in the Msx1 mutant, the dorsal midline of the diencephalon is affected and changes identity, correlatively with impairment of WNT1 signalling. In the Msx1: Msx2 double mutant, these defects extend to encompass the mesencephalon and frequently lead to exencephaly. On the contrary, the spinal cord is maintained and even contains extra cells. Preliminary analyses indicate that these cells are maintained because of a reduction in apoptosis. These results indicate that different mechanisms are involved in dorsal midline formation in the cephalic and spinal cord regions, and open the way to investigation of the identity and fate of roof plate cells.
Msx gene expression as a signature for cell plasticity (Olivier Goupille, Julie Moreau; in collaboration with Didier Montarras, Unit of Molecular Genetic of Development, and Ana Cumano, Unit of Lymphocyte Development)
During development, Msx genes are expressed, at induction sites, in mesenchymal cells that proliferate and differentiate into several histological cell types (bone, tendon, dermis...). Furthermore, their expression is induced in regeneration blastema in urodele amphibians and fish. These observations suggest that expression of Msx genes is associated with a state of cell plasticity characteristic of pluripotent stem cells. Taking advantage of the nLacZ reporter in the Msx1 and Msx2 loci, we have observed expression of these genes in scattered cells in adult mice, especially in blood vessels and in the dorsal midline of the neural tube. In blood vessels, expression is strictly regionalised along the vascular tree (photo 1) and restricted to a subpopulation of smooth muscle cells. We have produced, with the help of the Mouse Genetics Engineering Centre of the Institut Pasteur, a strain of mice that express GFP under the control of the Msx2 gene. These will permit to FACS -isolate Msx2-positive vascular cells and study their properties, with regard to differentiation and plasticity. This project is developed in the frame of the Strategic Horizontal Program (GPH) on "Stem Cells" of the Institut Pasteur.
Photo: Msx gene expression in the vascular tree of an adult mouse.
a) Msx2 is prominently expressed in the wall of peripheral arteries (here, femoral) of adult mice (arrow). Expression exhibits a helical pattern characteristic of smooth muscle cells. It presents with a punctuated pattern in the adjacent vein (arrowhead). b) Msx1 is expressed to a lesser extent in the femoral artery (arrow), but strongly in arterioles where it might label pericytes (arrowheads).
Keywords: Embryonic induction, organogenesis, limb bud, central nervous system, intercellular signalling, vascular smooth muscle, stem cells