Unit: Morphogenesis Molecular Genetics
Director: ROBERT, Benoît
Our laboratory is involved in the study of embryonic induction in vertebrate development. Emphasis is put on the role of Msx1 and Msx2, , two homeobox genes that are prominently expressed at sites of induction, in signal transduction associated with several signalling pathways (BMP, WNT...). Our approach relies on mouse mutants we have produced for both genes. We also analyse the relationship between expression of these genes and cell plasticity, in the prospect of isolating pluripotent somatic stem cells.
The Morphogenesis molecular Genetics Laboratory is involved in the study of ecto-mesodermal induction mechanisms that are implicated in organogenesis, with special emphasis on limb initiation and morphogenesis and on formation of the dorsal midline of the neural tube, in vertebrates. This work relies mainly on the properties and function of Msx1 and Msx2. These are homeobox genes prominently expressed in regions of induction between ectoderm and mesoderm. Both genes have been inactivated by insertion of an nLacZ reporter gene in the coding sequence, which further permits to monitor gene expression. Msx1 mutants exhibit cranio-facial defects at birth in derivatives of the first branchial arches (maxilla, mandible, tooth buds) which lead to perinatal death; Msx2 mutants are viable and fertile, but their hair follicles are abnormal and they loose hair shortly after birth. These phenotypes confirm the implication of Msx genes in inductive signal transduction.
Function of Msx genes in limb morphogenesis (Yvan Lallemand, Marie-Anne Nicola).
Neither Msx1 nor Msx2 mutation affect limb formation. However, the double homozygous mutant exhibits serious defects. Limbs are shorter and lack anterior skeletal elements (such as radius or tibia). In the autopod, the situation is more complex: usually, anterior elements (thumb) are missing, but often a paradoxical polydactyly may be observed. At later stages, inderdigital webbing does not regress. We have deciphered this complex phenotype and conclude that most of these defects may be related to impairment in BMP4 signalling, which plays several roles in limb morphogenesis. At early stages, BMP4 signalling contributes to setting the dorso-ventral polarity, which is necessary for apical ectodermal ridge formation and proximo-distal growth and patterning. In the Msx1:Msx2 mutant, the apical ridge does not form anteriorly, which explains anterior deficiencies. Later on, BMP4 constitutes the signal that stops the activity of the apical ectodermal ridge. This explains, at least partially, the overgrowth of the autopod and the polydactyly in the double mutant. Our analysis of Msx gene mutations contributes to the integration of signalling activities in limb formation.
Function of Msx genes in the formation of the dorsal midline of the neural tube (Antoine Bach, Yvan Lallemand, Mathilde Maufras).
Msx1 and Msx2, as well as Msx3, are also expressed at the dorsal midline of the neural tube. We have observed that in the Msx1 mutants, the subcommissural organ, an ependymal secretory structure located under the posterior commissure of the diencephalon, is absent, leading to hydrocephalus. Further investigation demonstrated that the whole roof of the diencephalon is affected. In the absence of Msx1, expression of several signalling factors normally expressed in the dorsal midline (BMPs, WNTs, ...) is lost, which leads to the downregulation of genes expressed laterally. Thus, Msx1 seems to be required for the function of the dorsal midline as a signalling centre. Analysis of the early phenotype and of Msx1/Msx2 double mutants shows that these genes are required for the expression of Wnt1, which plays an essential role in cellular proliferation in the dorsal neural tube. Our observations indicate that at this site, Msx genes may be involved in signal transduction between the ectoderm and the neuroectoderm.
MSX are transcription factors that bear a binding site for Groucho, a co-repressor in transcription. We have shown that MSX1 interacts with GRG3, one of the Groucho homologues in the mouse. Preliminary analysis suggests that the binding site for Groucho is required for induction of Wnt1 expression in the neural tube, and that MSX1 must interact with one of the GRGs for this function.
Expression of Msx genes as a signature of cell plasticity (Marie-Anne Nicola; in collaboration with Didier Montarras, Unité de Génétique moléculaire du Développement).
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 into differentiated myotubes leads them to fragmentation and re-entry in cell cycle. These cells can then give rise to derivatives from several lineages. These properties suggest that expression of Msx genes may be 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. Vascular cells in culture do proliferate while retaining Msx gene expression and simultaneously express markers from two distinct lineages, endothelium and smooth muscle. This work is pursued in the frame of a Strategic Horizontal Program (GPH) on stem cells that has been recently launched at the Institut Pasteur.
Keywords: Embryonic induction, organogenesis, central nervous system, intercellular signalling, hydrocephalus, cell plasticity