We are focusing on the mechanisms that link patterning and morphogenesis using mouse limb as a model system, and investigating the role of Msx1 and Msx2 homeobox genes in this process. We are further interested in cell fate acquisition in which these genes are involved, which extends our approaches to the CNS and the vascular system. We have generated several mutant alleles for Msx1 and Msx2 in the mouse, which constitute very efficient tools to study gene interactions and function.

Limb morphogenesis

The Msx2; Msx2 double mutant displays a complex limb phenotype. In particular, it is characterized by early defects that prevent, before E10.5, formation of limb anterior structures and preclude further analysis of this region. We have taken advantage of an Msx2 conditional allele we constructed to specifically abolish Msx function in the limb mesenchyme. This allowed us to obviate early defects. We thus demonstrated that Msx genes are required to prevent formation of an ectopic ZPA anteriorly. In the limb, antero-posterior polarization depends on the Shh / Gli3 signaling. Our preliminary results suggest that Msx synergize with Gli3R, and possibly Etv5, Twist1 and Hand2, in the anterior limb mesenchyme. We are furthering the investigation of these interactions by genetic and molecular analyses, to shed light on the mechansims of specification of the anterior region in the limb bud.

Role of Msx genes in the formation of the dorsal midline (roof plate) of the neural tube

Msx1 and Msx2 are expressed in the dorsal midline of the embryonic neural tube. In the spinal cord of Msx1; Msx2 double mutants, expression of Atoh1 is abrogated. Correlatively, the progenitors of the dorsalmost interneurons (dI1) are missing, and the whole dorsal region of the spinal cord is reorganized. This shows that Msx genes play a critical role downstream of the roof plate signals to specify dorsal interneurons. We are currently analysing the consequences of the inactivation of Msx genes on embryonic spinal cord patterning, and dissecting the spatiotemporal transcriptional network that leads from undifferentiated neural progenitors to mature neurons, in particular by the identification of Msx gene targets.

Mural cell recruitment and endothelium maturation in blood vessels

We showed that in adult mice, Msx1 and Msx2 are expressed in the smooth muscle layer of peripheral arteries, up to the latest stages of life. Msx1 expression is further observed in a sub-population of pericytes in capillaries. In the embryo, we have further demonstrated that mutation of both Msx1 and Msx2 leads to a decrease in smooth muscle coverage of the head arteries. This results in increased vessel diameter, excessive branching and impairment of endothelium layer maturation, which might be of relevance to human pathology. Using an Msx1Cre-inducible allele we have recently produced, we could identify a population of vascular smooth muscle precursor cells in which Msx genes are required. In the mutant, these cells cannot migrate properly to their differentiation site.

Keywords: embryonic induction, organogenesis, limb bud, central nervous system, intercellular signaling, vascular smooth muscle, progenitors

Lallemand Y, Bensoussan V, Saint Cloment C, Robert B. 2009. Msx genes are important apoptosis effectors downstream of the Shh/Gli3 pathway in the limb. Dev Biol 331:189-198. PMID : 19422820

Goupille, O, Saint Cloment, C, Lopes, M, Montarras, D, Robert, B. 2008. Msx1 and Msx2 are expressed in sub-populations of vascular smooth muscle cells. Dev Dyn 237:2187-2194. PMID : 18627106

Hu, J. S., Doan, L. T., Currle, D. S., Paff, M., Rheem, J. Y., Schreyer, R., Robert, B. and Monuki, E. S. 2008. Border formation in a Bmp gradient reduced to single dissociated cells. Proc Natl Acad Sci U S A105 :3398-403. PMID: 18292231

Robert, B. 2007. Bone morphogenetic protein signaling in limb outgrowth and patterning. Dev Growth Differ 49:455-468. PMID : 17661740