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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. Genetic tools for molecular imaging are being engineered to analyze in transgenic animals the functional synaptic organization of the brain during embryogenesis and later during activity-dependent synaptic rearrangement. Anterograde and retrograde sigalling are essential for the development maintenance and activity-dependent modification of synaptic connections.



I - Genetic analysis of neural network functioning in the mouse developing brain

To assess connectivity fusion proteins between tetanus toxin fragments and a reporter gene LacZ or GFP were constructed. These latter molecules retain the holotoxin intracellular and trans-synaptic transport properties and will therefore report on retrograde signalling in neural networks. Various forms of retrograde signalling have been identified at developing and mature synapses. Pre-synaptic functions are regulated by retrograde signals in an activity dependent manner with a rapide time course. Long range pre-synaptic propagation of the retrograde signals occur in some cases, conveying regulatory signals to the nucleus and affecting functions in distant parts of the neuron. In this context, we are caracterizing the retrograde and trans-synaptic transport detected by our hybrid probes, at the neuromuscular junction and during later processes in the developing CNS. Informations will also be gained on how toxins enter and propagate inside the nervous system.

a) The neuromuscular junction (Sylvie Roux, Cécile Saint Cloment)

The TTC hybrid proteins have been intramuscularly injected to analyze in vivo the mechanisms of intracellular and transcellular traffics at the neuromuscular junction (NMJ). In muscle, a directional membrane traffic concentrates b gal-TTC hybrid protein into the NMJ. 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 strongly dependent upon the pre-synaptic neural cell activity. Molecular details of b gal-TTC inter and intracellular transports have been studied by confocal and electron microscopy. The hybrid proteins bind to their membrane receptors, gangliosides GT1b and GD1b and to a GPi linked protein p15, are internalized inside caveolae-like vesicles and transported to various intracellular compartments into motoneuron, muscle and NMJ. We are postulating that structural and dynamical properties of lipids microdomains in a post-synaptic cell are modulated by a pre-synaptic neural cell activity, leading to their rapid clustering to an active synapse. We are analyzing the molecular and cellular details of that dynamical process. As an internal control, cholera toxin B chain has been co-injected and displays a different distribution and traffic. Cholera toxin receptor differs from TTC receptors. We are therefore detecting a precise adressing of membrane microdomains to different intracellular organelles.

b) Caracterization of retrograde pathways (Sylvie Roux, Cendra Agulhon, Rafael Vazquez-Martinez)

GFP-TTC allows us to visualize a retrograde membrane traffic from the outputs of a neuron to its inputs as well as a trans-synaptic transport of molecules. Throughout evolution tetanus toxin have parasitized a constitutive vesicular transport to invade the CNS deeper layers. Such a constitutive transcytotic membrane traffic retrogradely links an active axonic junction to an active dendritic synapse. We postulate that such a traffic could be involved in adjusting the neural cell physiology inside its active network. To establish its biological role requires the identification of the molecules that are on the same rafts and vesicles, being co-transported together with TTC receptors. To correlate retrograde signalling via membrane transport to the post-synaptic cell physiology, electrophysiological stimulation are applied to pre-synaptic cells within organotypic slides of neural tissue.

c) Genetic analysis in transgenic animals (Sandrine Picaud, Thomas Curie, Sylvie Roux, Rafael Vazquez-Martinez, Cécile Saint Cloment, Cendra Agulhon)

Simple transgenic animals expressing GFP-TTC have already been constructed and analyzed. Cell specific promotors like calbindin, as well as ubiquitous promotor, CMV, have been used. Our results in vivo have established the approach's feasibility using a combination of two reporter genes. Likewise, we can identify during embryogenesis cells in which transcription occurs and connected cells receiving a reporter. The dynamic progression of the reporter protein inside the neural network as well as the details of the intracellular transport can be monitored by multiphotons confocal microscopy, eventually when possible in living animals.

d) Imaging neuronal activities in networks (Kelly Rogers)

Calcium is an universal second messenger with critical roles at various levels of neural information processing. Developmental changes in cell-cell interactions may also be monitored by calcium imaging. 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 powerful imaging system allows us to investigate calcium activities with greater temporal and spatial resolution in neuronal networks.

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

In the last few years, live births have been achieved using somatic nuclear transfer in various mammals. The overall effect of transferring a somatic nuclei into an egg is to reboot its genetic program for embryogenesis. Besides, fusion of an embryonic stem (ES) cell with a somatic cell produces an hybrid cell with totipotency properties. Yet unidentified factors localized into the egg cytoplasm or expressed in ES cells can reprogram genes in a coordinated fashion. This phenomenom also occurs naturally, for example in the amphibian Urodele when a limb is severed. Somatic cells enter dedifferentiation then proliferate and form new tissues. 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.

We have constructed a new retrovirus vector designed for an exhaustive mutagenesis of a somatic cell genome in vitro. The loxP/Cre recombinase system is used to eliminate most viral DNA after integration. The remaining single LTR, which comprises strong enhancers, will transcribe the gene downstream of the integration site. A selectable phenotype should be provided by the expression of a marker specific for the undifferentiated totipotent state. The transcription factor Oct-4 is specifically expressed in totipotent cells of mouse embryos according to a dual regulation in germ and somatic lineages. A distal enhancer (DE) region is responsible for expression in the pre-implantation embryo, the germ line and ES cells while a proximal enhancer (PE) drives Oct-4 expression in the epiblast and embryonal carcinoma (EC) cells. Expression of a GFP-cell surface marker under the control of Oct-4 distal enhancer would allow the selection of an ES cell-like phenotype by using the powerful magnetic cell sorting. To perform the mutagenesis, a transgenic animal would be the most convenient source of all kinds of tissues at every developmental stage.

Photo: GFP-TTC distribution in dendritic spines of a pyramidal cortical neuron in culture. Transfection was achieved by incubation with FUGENE and pCMV-GFP-TTC for 48 h.

Keywords: Developmental neurobiology, de-differentiation, synaptic organization, calcium imaging, homeogene, transgenesis, stem cells


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  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

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

ROUX Sylvie (PhD) : sroux@pasteur.fr

ROGERS Kelly (PhD) : krogers@pasteur.fr

AGULHON Cendra (PhD) : agulhon@pasteur.fr

VAZQUEZ-MARTINEZ Rafael (PhD) : rvazquez@pasteur.fr

CIRIZA-ASTRAIN Jesus (trainee) : jciriza@pasteur.fr

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

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

RUSSE Sophie (IP) : srusse@pasteur.fr

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