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 in vertebrates.
Functional analysis of Msx genes in the mouse (Antoine Bach, Marie-Anne Nicola, Yvan Lallemand). Msx1 and Msx2 are homeobox genes prominently expressed in regions of induction between ectoderm and mesoderm. Msx1 was inactivated a few years ago by insertion of an nLacZ reporter gene in the coding sequence. Mutants exhibit cranio-facial defects at birth in derivatives of the first branchial arches (maxilla, mandible, tooth buds) which lead to perinatal death. We further observed that they suffer from hydrocephaly. This new phenotype appears to be due to a deficiency in the formation of the subcommissural organ, an ependymal secretory structure located under the posterior commissure of the brain. In fact, the whole roof of the diencephalon seems affected (fig. 1). We are currently investigating the interference of the Msx1 mutation with signalling pathways identified at the level of the fore-midbrain junction (BMPs, Wnts, FGFs
We have achieved the mutation of Msx2 by homologous recombination. The whole coding sequence of the gene was replaced by an nLacZ reporter gene. Using this marker, we are revisiting the expression profile of Msx2 with increased sensitivity and accuracy. We previously characterised expression of Msx1 in progenitor cells of the dermis that migrate out of the somite dermomyotome. No alteration was observed at this site in the Msx1 mutant, but redundancy with Msx2 is possible. Actually, we observe a segmental, dorsal expression of this gene which partially overlap with that of Msx1. Then, the Msx1/Msx2 double mutants will be instrumental in understanding the function of Msx genes in development.
Relations between BMP signalling and Msx genes (Ewa Szabat, Marie-Anne Nicola). In a number of sites in the embryo, there is a strong correlation between Msx1 or 2 and Bmp2/4 expression patterns. We have undertaken the controlled ablation of BMP2/4 activity in mouse embryo by conditional expression of noggin, a potent inhibitor of these BMPs, using tetracycline-inducible promoters. This is achieved by associating two transgenes, one expressing a transcriptional activator, rtTA, which is active only in the presence of tetracycline; the other expressing noggin cDNA under the control of a tetO operator, which is activated by rtTA. Association of the two transgenes lead to tetracycline dependence for noggin expression. Vectors have been constructed for these transgenes and transfected in ES cells. Twelve independent clones were selected that will be used to derived mouse strains.
In addition, we are analysing the transactivation of BMP4 promoter (which contains binding sites for the MSX) by MSX1 and MSX2 proteins, using cell culture systems.
Analysis of mouse polydactylous mutants (Isabelle Blanc). We are studying a new mouse polydactyly, Pluridigité (Pdt). The phenotype appears to depend on the interactions between three genes, a major locus on chromosome 12, close to Twist, and two strong modifier loci on chromosome 4. These mutations have been fixed in a recombinant inbred strain, which constitutes a precious tool to investigate interactions between the three genes.
Relations between apoptosis and morphogenesis (André Weydert). Protein phosphatase 2A (PP2A) is involved in numerous activities in cell metabolism, including apoptosis. We have undertaken the in situ inhibition of PP2A in the chick wing bud by implanting inhibitor-loaded beads. This leads to specific and reproducible limb agenesis which follows the presumptive maps of the wing. Intercellular signalling mechanisms which may be affected in this process are under study.
Fig. 1: At E14.5, the diencephalon midline in an Msx1 mutant (A) does not express Msx1. Cells which normally express this gene (B) either have been eliminated, or have changed fate.
Fig. 2: Msx2, as Msx1, is expressed in dorsal demis progenitors at E12.5 (arrows).