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Homeogenes and adhesion molecules in the nervous system

Objective

The objective is to understand the roles and modes of action of homeodomain proteins and adhesion molecules in the development of the nervous system.
Analysis of otx-2 expression during development. Studies on the evolution of the cortex with molecular markers. Molecular and functional dissection (in vitro and in vivo) of 5'upstream regions of several adhesion molecules. Analysis of the compartimentation of adhesion molecules and transcription factors and of their mode of action. Generation of sevaral knock-out and analysis of phox-2 and Ncam knock-out phenotype. Analysis of neuronal plasticity in the adult.

MAJOR SCIENTIFIC BREAKTHROUGHS:
Laboratory 1 has shown that the Amyloid Precursor Protein (APP) has a role in neurite elongation and that axons contain a small pool of APP wich is restricted to microdomains with caveolar properties containing F3/F11 (collaboration with laboratory 7). With laboratory 9, laboratory 1 has introduced a peptidic PKC inhibitor in growth cones and shown that PKC inhibition in live cells provokes growth cone collapse. The introduction of the same peptide in neurons does not modify their response to tenascin-R (collaboration with laboratory 8). Laboratory 2, with laboratory 10, has studied the expression chick-otx2 in the developing embryo. c-Otx2 is first expressed in the entire epiblast and in the hypoblast and mesoblast with a progressive restriction to the Hensen's node. In a second phase c-Otx2 is found in the anterior neurectoderm and mesendoderm, notochor and floor plate excepted. Compared to gsc, c-otx2 is transcribed first in the hypoblast. The expression domains of the two genes transiently overlap in Hensen's node, gsc being an earlier marker of the primitive streak. Only c-otx2 is expressed in neuroectodermal areas. The second phase of c-otx2 expression is sensitive to an early treatment with retinoic acid. Laboratory 3 has characterized with laboratory 4 the 5' upstream region of F3/F11. Constructs in which distinct elements control a reporter gene expressed in transgenic animals demonstrate a specific expression in CNS and PNS neurons. A neural enhancer region has been characterized as well as cAMP and retinoic acid responsive elements. Two nuclear factors that bind distinct regions in the F3 promoter have been characterized. With laboratory 7, a chimeric proteins encompassing F3/F11 and NCAM Ig domains 4 and 5 were produced. This chimeric protein is not polysialylated on Ig domain 5 whereas the VASE exon in the Ig domain 4 still regulates neurite outgrowth. The two laboratories have produced soluble F3 to identified new F3/F11 molecular targets. Laboratory 4 has shown that the expression of Phox2 is limited to parts of the autonomic nervous system and to the adrenergic nuclei in the brainstem. In the sympathoadrenal lineage and some of the cranial ganglia Phox2 is expressed at the onset of adrenergic differentiation. Phox2 knock-out mice have been produced. In the mutants, parasympathetic ganglia, the Phox2-positive cranial ganglia and the locus coeruleus are affected. In the locus coeruleus, DBH and TH are never expressed. The cranial ganglia expressing Phox2 form correctly, but fail to express c-ret and start degenerating around mid-gestation, possibly because of the absence of c-ret. The NCAM knock-out phenotype has been studied in collaboration with laboratories 4 and 7 (effects on neurite outgrowth) and 8 (behaviour, see below). Double mutants for MAG and NCAM generated with laboratory 8 demonstrate that MAG and NCAM act synergistically to alterate myelin formation. Laboratory 5 has worked with laboratory 3 on the role of TAG-1 and F3 in vivo using transgenic models, with laboratory 9 on the regulation of TAG-1 during degeneration and regeneration and with laboratory 10 on the migratory pathways in the olivocerebellar system using a number of markers, including TAG-1. The human homologue of TAG1 was characterized and localized on chromosome 1q32. The expression pattern of TAG-1 in the human embryo is under study. Soluble chimeric forms of human TAG-1 and also of Ig or FN domains have been produced. Laboratory 6 has collaborated with most other laboratories to identify the regions of the central nervous system where several genes studied in this grouping are expressed. In collaboration with laboratory 10, laboratory 6 has pursued the study of an organizing region at the mesencephalic-metencephalic border and demonstrated an important role for FGF-8. Laboratory 7 has prepared F3/NCAM chimeric molecules to examine their effects on neurite outgrowth (collaboration with laboratories 3 and 4). The VASE exon on NCAM is able to work as a modular motif. With laboratory 3, it was demonstrated that neurite elongation is inhibited (cerebellar granule cells), stimulated (sensory ganglions) or not affected (hippocampal neurons) by F3 Ig domains. With antibodies provided by laboratory 5, laboratory 7 is doing a structure-function analysis and identifying the receptors for TAG-1 and F3 on cerebellar granule cells. With laboratory 8, laboratory 7 has demonstrated that F3 associates with L1 and Fyn tyrosine kinase within small cerebellar membrane microdomains. The tenascin-R binding site on F3 has been mapped and MAG has been identified as another ligand of tenascin-R. Laboratory 8, with laboratory 4, has worked on the behaviour of mice genetically deprived of NCAM and demonstrated a modification in male aggressivity (correlated with changes in neuroendocrine activity) and in long term potentiation. With laboratory 3, laboratory 8 has studied the L1 promoter and brought significant evidence for a regulation of promoter activity by homeoprotein pax-6 and possibly by homeoprotein pax-8. Laboratory 9 has studied the molecular basis of neuronal plasticity in the adult. MAP1B-P expression largely co-localizes with PSA-NCAM and is maintained in several region in particular those showing plasticity in the adult (e.g. olfactory system, hippocampus, some hypothalamic nuclei). With laboratory 7 the time course of expression of these markers was followed in the magnocellular nuclei of the hypothalamus. PSA-NCAM levels progressively decrease in oxytocinergic neurones to become almost undetectable at the end of the second weeks of lactation. In the same conditions, MAP1B-P is significantly up-regulated in post-hypophysis, the projection territory of oxytocin neurones. The two markers returned to control levels after weaning. With laboratory 2, laboratory 9 has investigated the possible role of the Otx-2 homeoprotein in regulating the expression of cell-surface molecules during development of the CNS. Several targets of the retina strongly express Otx2. Otx2 was transfected in NIH3T3 cells, and neurite outgrowth was tested on membrane prepared from control or transfected cells. Retinal neurites are longer and more numerous on membranes of transfected cells. N-CAM 140 and Thy-1 are regulated in correlation with the level of Otx2 expression. Laboratory 10 has worked with laboratory 2 on the expression of chick-otx-2 (see above) and compared the expression of molecular markers of telencephalic regionalization (emx1, dlx1, pax6) in a bird, a reptile and a mammal. Contrary to a widely accepted opinion, it was shown that the so-called Dorsal Ventricular Ridge is not embryologically related to the neocortex but to a discontinuous ring of early generated mammalian nuclei. In collaborarion with laboratory 5, laboratory 10 has studied the migration of inferior olivary cells in vitro.

Funding Scheme

CON - Coordination of research actions

Coordinator

ECOLE NORMALE SUPERIEURE
Address
Rue D'ulm 45
75230 Paris
France

Participants (9)

Centre National de la Recherche Scientifique (CNRS)
France
Address
1 Avenue De La Terrasse
91198 Gif-sur-yvette
FOUNDATION FOR RESEARCH AND TECHNOLOGY -HELLAS
Greece
Address
Matzapetaki 21
71110 Heraklion-crete
Institut National de la Santé et de la Recherche Médicale (INSERM)
France
Address
47 Boulevard De L'hôpital
75651 Paris
Istituto Scientifico ' San Raffaele '
Italy
Address
Via Olgettina 60
20132 Milano
Istituto di Fisiologio Umana
Italy
Address
Giulio Cesare 1
70124 Bari
MOLECULAR BIOLOGY OF CELL ADHESION
France
Address
Luminy
13288 Marseille
SWISS FEDERAL INSTITUTE OF TECHNOLOGY
Switzerland
Address

8093 Zurich
UNIVERSIDAD DE MURCIA
Spain
Address
Campus Murcia
30100 Murcia
Université d'Aix-Marseille II
France
Address
70 Route Léon Lachamp
13288 Marseille