Interaction between guidepost cells of the corpus callosum and callosal axons
To test whether neurons of the corpus callosum attract or repel callosal axons and whether they exert short or long range distance actions, we are currently using in vivo model of transgenic mice, co-culture explants as well as our in vitro model of corpus callosum organotypic slices in which the organization of the corpus callosum and the cortical projections is preserved. We are testing if neurons of the corpus callosum are required for the normal growth of cortical axons with loss-of-function experiments. We are also testing whether corpus callosum neurons facilitate the growth of callosal axons in an otherwise non permissive environment. Finally, in order to establish definitively the involvement of neurons in the guidance of callosal axons, we are testing whether transplantation of wild-type corpus callosum neurons can restore callosal pathfinding using neurons-free mutant slices. To gain insight into how growth cones of callosal axons respond to neurons and glia of the corpus callosum along their pathway, we are using confocal time-lapse video microscopy to monitor the dynamic behaviors of individual callosal growth cones extending into organotypic corpus callosum slices. Our study will allow us to determine whether the complex callosal growth cone behaviors correspond to interaction with neuronal and/or glial cellular targets.
Mechanisms by which neurons guide commissural axons
In parallel experiments, we are investigating the molecular mechanisms of guidance by studying if guidepost neuronal cells operate to direct commissural axons outgrowth by secreting guidance factors or by expressing adhesion molecules. We are examining the expression profile of corpus callosum neurons for guidance cues known to regulate pathfinding. This includes secreted guidance factors (Netrin, Slit, Semaphorins and several ephrins and their receptors) as well as adhesion molecules. To determine whether the identified genes are important for commissural axons pathfinding, we are analysing mice carrying a specific inactivation of the gene of interest or mice with the specific gene inactivated by RNAi knockdown after in utero electroporations (see "technologies" part on the DBCM website). The specific responses of callosal axons to these molecules are studied by looking at the growing callosal axons in different guidance assays: (1) cortical explants placed at a distance from COS cell aggregates expressing the molecule of interest (2) aggregates of expressing COS cells placed in the corpus callosum slice. We are studying the reaction and dynamic behaviour of callosal axons while they encounter the aggregates with confocal time-lapse video microscope as well as in fixed tissues. We are applying this strategy to test the cellular response of callosal axons to well characterized guidance molecules as well as molecules we have identify by screening with microarrays or PCR.
Origin and regulation of astroglial cell specification in embryonic brain
Part of our project consists in characterizing the origin of astroglial cells in embryonic brain as well as the contribution of various genes in regulating astroglial cell specification. The brain tissue is made of neuronal and glial cells generated in the germinative layer bordering the ventricules deep in the neuronal tube. These cells divide, differentiate and migrate following specific pathways. The cell fate of glial progenitors are traced by using GFP reporter mouse line mice. The origin of astroglial cells is directly visualized by combining glial immunostaining and electroporation (see "technologies" part on the DBCM website) into various ventricular zone subdomains. To provide further indications on the brain region generating early astroglial cells, domains are dissected out, cultured separately and domain explants expressing astroglial cell clusters are identified. To gain insight into the specific migratory routes and dynamics of astroglial precursors from their site of origin toward the brain midline, we are using confocal time-lapse video microscopy on transgenic brain slices. We aim to determine the genes that are required for the generation of astroglial precursors or to specify the astroglial identity of the generated precursors. The functions of these candidate genes in cell fate decisions is directly addressed in experiments with neurospheres, as well as, in vivo.
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A full publication record can be found here.