Unit: Morphogenesis Molecular Genetics
Director: Benoît ROBERT
Our laboratory is studying the function of Msx homeobox genes, using mouse mutants that we 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. Experimental evidence further suggests that they play a role in maintenance of cell plasticity. Through the analysis of these genes, we are involved in the study of fundamental mechanisms of embryonic induction and of stem cell properties.
The laboratory of Molecular Genetics of Morphogenesis is involved in studying 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 allowed extending considerably these analyses. These mutants confirm the implication of Msx genes in inductive signal transduction required for organogenesis particularly in limb outgrowth and morphogenesis, and in the formation of the dorsal midline of the neural tube, in vertebrates.
Role of Msx genes in limb morphogenesis (Yvan Lallemand, Marie-Anne Nicola)
Contrary to what is observed in either Msx1 or Msx2 simple mutants, the homozygous double mutant exhibit severe defects in limbs. 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, cases of polydactyly are not rare (photo1). 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, BMP4 is the signal that switches off the apical ridge activity and distal proliferation of the limb bud. Due to lack of BMP signalling, FGF activity is maintained, apoptosis is abolished, which lead to outgrowth of the autopod and formation of anterior polydactyly. Extra tissues nonetheless keep a posterior identity, demonstrating that they derive from a limb bud initially truncated. This work shows the importance of Msx genes in BMP signal transduction, in the anterior region of the limb bud as at other sites (such as teeth).
Role of Msx genes in the formation of the dorsal midline of the neural tube (Valérie Borel, Yvan Lallemand, Marie-Anne Nicola)
Msx1 and Msx2, as well as Msx3, are also expressed in the dorsal midline of the neural tube. 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 of apoptosis. Msx genes would thus be required for apoptosis at this site. Using the nlacZ reporter gene, we can follow the fate of midline cells, which have been poorly described, and analyse the role of Msx genes in the formation, maintenance and functional activity of the midline.
Msx gene expression as a signature for cell plasticity (Olivier Goupille; 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. Recent work has shown that forced expression of Msx1 into differentiated myotubes leads them to break up and re-enter cell cycle. Proliferating cells can then give rise to derivatives from several lineages. 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. By clonal analysis, we have shown that single vascular cells give rise to cell populations which simultaneously express markers from two distinct lineages, endothelium and smooth muscle. This implies that they are pluripotent. This work is developed in the frame of a Strategic Horizontal Program (GPH) on stem cells that was recently launched at the Institut Pasteur.
Photo : Posterior limb buds from (1, 2) a double homozygous mutant for Msx1:Msx2 (DH) or (3) from a normal embryo at 12.5 days of development. A single embryo may exhibit both a polydactyly (1, left limb, 6 digit anlagen) and an oligodactyly (2, right bud, 4 anlagen). Polydactyly is always on the anterior side (A). Note that mutant limbs are smaller, digit cartilages are poorly individualised and in most cases, the tibia is missing (2, asterisk).
Keywords: Embryonic induction, organogenesis, limb bud, central nervous system, intercellular signalling, cell plasticity, stem cells