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  Director : Philippe BRÛLET (pbrulet@pasteur.fr)



The precision with which neuronal circuits are assembled during development is critical in defining the behavioral repertoire of the mature organism. The genetic specification and functioning of neural circuits in the developing mouse embryo are the focus of our research. The roles of homeogenes Otx and Emx as well as FGF-15, a secreted factor are particularly studied in the synaptic organization of the brain. Genetic tools for molecular imaging are being developed to analyze in transgenic animals neural networks activity and connectivity during their establishment in embryogenesis, during their refinement and during a learning paradigm.



Genetic analysis of neural networks connectivity in transgenic animals (S. Roux, C. Saint Cloment, T. Curie, J. Miana-Mena)

We have constructed several fusion proteins between tetanos toxin fragments and a reporter gene, LacZ or GFP. These molecules retains retrograde intracellular and trans-synaptic transports properties. The same hybrid proteins have been intramuscularly injected to analyze in vivo the mechanisms of intracellular and transcellular traffics at the neuromusclar junction (NMJ). In muscle, a directional membrane traffic concentrates b gal-TTC hybrid protein into the NMJ post-synaptic side. In neuron, the probe is sorted across the cell to dendrites and subsequently to an interconnected neuron. Traffics on both sides of the synapse are therefore strongly dependent upon the presynaptic neural cell activity. Simple transgenics animals have been constructed and analysed. Cell specific promotors like calbindin, as well as an ubiquitous promotor, CMV, have been used. Our results in vivo have established the approach's feasibility using a combination of two reporter genes. We can identify during embryogenesis cells in which transcription occurs and connected cells receiving reporter proteins by an activity dependent transport .We are analyzing connectivity in various genotypic context by constructing animals with a genetic conditional triggering of the reporter genes synthesis. The dynamic progression of the reporter protein inside the neural network as well as the details of the intracellular transport will be monitored by multiphotons confocal microscopy, eventually when possible in living animals.

Calcium imaging in neural networks (V. Baubet)

Calcium is an universal second messenger with critical roles at various levels of neural information processing. By analogy with the fluorescing jellyfish in response to calcium influx, we have constructed new calcium sensitive bioluminescent proteins by fusing aequorin and GFP . The main advantage of this new calcium imaging is that we can target the gene to intracellular organelles, to specific receptors and channels, as well as specific neural subpopulations in transgenics animals. In addition, optical microscopy allows to detect long range correlation of calcium fluctuations in different cells of a tissue. We have already targeted such a reporter gene to the Hoxc-8 locus in a transgenic animal and to the pre-synaptic apparatus by fusion with synaptotagmin. We are planning to target the reporter protein to the endoplasmic reticulum and to active dendrites so as to follow, in transgenics animals, the intracellular modifications of neuronal informations during its processing and successive integration from a dendritic compartment. A new and powerful imaging system will be used to follow in real time calcium fluctuations.

Genetic mechanims of brain development (R. Hashemi, S. Picaud)

Null mutant mice with Otx1 or Otx2 homeogenes replaced by LacZ were created. By 9.5 dpc homozygous Otx2-/-mutant embryos are characterized by the lack of brain structures anterior to rhombomere 3. Otx2 expression at the onset of gastrulation is required for neural induction. Gene expression profiles in WT and mutant embryos were compared by SAGE at E6.5. Among a broader list , the study of Otx2 downstream genes allows defining a role for Otx2 in the orchestration of cell movements leading to the adequate organization of the gastrulating embryos. One likely Otx2 downstream gene is the secreted factor FGF-15 ; It is expressed in a gradient fashion along the proximo-distal axis only in ectodermal cells at E 6.5. Aside from this early role, FGF-15 is transcribed in a regionally-restricted pattern in the developing nervous system, particularly within the Isthmus and the Zona Limitans Intrathalamicus - ZLI. Its conditional mutagenesis is undertaken. Otx1-/- mice show a later phenotype,epileptic behavior and multiple brain abnormalities. Otx1 mutant mice are also defective in the refinement of long distance, exuberant axonal projections, suggesting that Otx1 is required for the development of normal axonal connectivity and the generation of coordinated motor behavior .

Reprogramming somatic cells into stem cells (H. Le Mouellic)

In the last few years, live births have been achieved using somatic nuclear transfer in mice, sheep, cows and goats and the technique would probably also be successful in human. The greatest benefit of the new technology will likely be in therapeutic cloning ; the use of somatic cell nuclear transfer to generate replacement tissues or organs. This would avoid the risks of tissue rejection by supplying a person with new tissues of exactly their own genetic type. The overall effect of transferring a somatic nuclei into an egg is to reboot its genetic program for embryogenesis. Yet unidentified factors localized into the egg cytoplasm can reprogram genes in a coordinated fashion. Knowing that a developmental program can be rebooted, we explore if one can genetically reprogram somatic cells into stem cells and identify the factors involved. A new genetic method relying on an exhaustive mutagenesis in vitro is being tested.


puce Publications of the unit on Pasteur's references database


  Office staff Researchers Scientific trainees Other personnel

DE GROOTE Dominique (IP – Secrétaire) : degroote@pasteur.fr

BRULET Philippe (CNRS/IP - Responsable Unité) : pbrulet@pasteur.fr

LE MOUELLIC Hervé (Inserm) : hervelm@pasteur.fr

BAUBET Valérie (Post-doc) : departure 12.31.01

CURIE Thomas (PhD student) : tcurie@pasteur.fr

HASHEMI Reza (PhD student) : rhashemi@pasteur.fr

MIANA MENA Javier (trainee) : jmiana@pasteur.fr

PICAUD Sandrine (CNRS - Engineer) : spicaud@pasteur.fr

ROUX Sylvie (Post-doc) : sroux@pasteur.fr

SAINT CLOMENT Cécile (IP - Technician) : ccloment@pasteur.fr

RUSSE Sophie (IP) : srusse@pasteur.fr


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