Unit: Molecular Embryology
Director: Philippe BRÛLET
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.
I - Genetic analysis of the functional synaptic organization 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 rapid 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 in the developing CNS. Information will also be gained on how toxins enter and propagate inside the nervous system. In an alternative approach, we are using the calcium reporter GFP-Aequorin to directly visualize the propagation of activity in neural cells networks.
a) The neuromuscular junction (Cécile Saint Cloment, collaboration with Sylvie Roux, Jordi Molgo - CNRS UPR 9040)
Intramuscularly injected hybrid protein will specifically bind to the active NMJ, be endocytosed and retrogradely transported to the soma, active dendrites and to interconnected neural cells. In vivo, this traffic is strongly dependent upon the pre-synaptic activity. Various neurotrophic factors modulate the transport kinetics. We postulate that several structural and dynamical properties of lipid microdomains in a post-synaptic synapse are modulated by pre-synaptic activity. Such traffic could be an integrative mechanism allowing a flow of retrograde information in an active neural network. To analyse the cellular and molecular mechanisms involved, experiments are being carried-out at the biochemical level but also by confocal and electron microscopy.
b) Characterization of TTC trafficking in central synapses (Rafaël Vazquez-Martinez)
The main goal of this project is to elucidate different cellular aspects of retrograde signaling in the central nervous system. We have therefore taken advantage of the characteristics of neurospecific binding and retrograde transport displayed by the non-toxic fragment of tetanus toxin (TTC) tagged with the green fluorescent protein (GFP). Throughout evolution, tetanus toxin has parasitized constitutive mechanisms of retrograde vesicular transport to be addressed towards specific structures ( i.e. dendritic spines where synaptic contacts are formed) and to trans-synaptically invade the pre-synaptic terminal. Interestingly, uptake and transcytosis of TTC appear to be membrane trafficking pathways regulated by a neurotrophic factor and key component of retrograde signaling, the brain-derived neurotrophic factor ( i.e. BDNF). We propose that these membrane trafficking pathways would participate in bringing in and out of the active synapse (at the pre-synaptic as well as the post-synaptic side) necessary components for modulating synapse activity. To ascertain this hypothesis, we are carrying out time-lapse visualization of pyramidal neurons in thick organotypic slices under different pharmacological as well as electrophysiological conditions. Variations in intracellular content of TTC (indirectly assessed by the fluctuactions of intracellular fluorescence) as well as morphological remodeling of dendritic spines over time induced by the different experimental protocols tested should provide important information on the cellular mechanisms underlying membrane trafficking at the active synapse.
c) Analysis of the functional synaptic organization in transgenic animals (Sandrine Picaud, Thomas Curie, Pierre Godement)
Transgenic animals expressing GFP-TTC have already been constructed and analyzed. Cell specific promotors like calbindin, as well as ubiquitous promotors, CMV, ROSA 26, have been used. Our results in vivo have established the feasibility of this approach 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 multiphoton confocal microscopy allowing to visualize the functional synaptic organization in mutant transgenics animals. In one transgenic animal, GFP-TTC is expressed in retinal cells and the visual system. Its expressions pattern is being established and functional experiments are being made to study the protein trans-synaptic transport.
d) Visualization of neuronal network activity using the bioluminescent calcium sensitive reporter GFP-aequorin (Kelly Rogers, Cendra Agulhon, Jacques Stinnakre, Sandrine Picaud)
We are developing an approach to visualize in real-time' functional neural network activity in tissue and whole animals using the Ca2+ reporter GFP-aequorin (GA). The fluorescence of GFP allows expression patterns of the Ca2+ reporter, aequorin, to be visualized. After Ca2+binding to aequorin, energy is transferred to GFP, whereby green light is emitted in a process known as chemiluminescence energy transfer (CRET). Overall, the stability of the protein is improved and light emission of the photoprotein is significantly higher than that produced by aequorin alone. In comparison to fluorescent probes, the Ca2+-induced bioluminescence produced by GFP-aequorin does not require light excitation. The reporter has an excellent signal to noise ratio and can be utilized for long-term imaging. We have shown that GFP-aequorin can be genetically targeted to different subcellular compartments in the pre- and post-synaptic apparatus. Transgenic animals have been constructed with several targeted probes in order to visualize different aspects of network activities at various developmental stages. In one animal, the probe is targeted to mitochondria. In a second animal, GA is fused to PSD.95 and is conditionally expressed in the dendritic synapse of pyramidal cells, in close proximity of the NMDA receptor. Spontaneous and rhythmic recurrent activities in neocortex will be analyzed during late embryogenesis and early adulthood in relation to critical periods. Particularly, we will test the balance between excitatory and inhibitory activities in large neural ensembles. GA transgenic mouse strains can be crossed with any brain patterning mutant animals as well as neurodegenerative disease model mice to follow perturbed neural calcium homeostasis. Finally, whole animal luminescence calcium imaging is being performed to image specifically targeted intracellular activities and analyze brain processes on a moving animal.
A virus expressing the bioluminescent GFP-aequorin probe is used to transfect mouse retinal organotypic slices. High level of infection specifically within glial cells but not in neurons is obtained. We show that the cytoplasmic GFP-aequorin is functional and stable. It allows to reveal the localization of calcium activity previously described in literature using fluorescent dyes and to detect several calcium waves after successive ATP stimulations. Finally, this new bioluminescent imaging technique enables detection of spontaneous calcium transients. In conclusion, GA allows to measure spontaneous and evoked activities of neural cells in tissue preparations. In addition, GA calcium sensitive protein could also represent the reporter of choice in studies on retinal biological model that are sensitive to light which should be carefully regulated.
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 pluripotency 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 transgenic mouse line containing a genetic marker of pluripotent cells. A minimal promoter has been associated to the enhancer sequences responsible of Oct-4 expression in Embryonic Stem (ES) cells. The reporter is a fusion protein exposing the GFP on the outer surface of cell membrane (photo). The transgene is specifically expressed in ES cells, pre-implantation embryos and the germ line of transgenic animals. Insertional mutagenesis with viral vectors could be performed to induce reprogrammation of totipotency in various cell populations obtained from this mice. The presence of several epitopes allows to isolate rare positive events using the powerful magnetic cell sorting.
photo 1: Localization of GFP-TTC tracer at the neuromuscular junction. After the deposit of the fusion protein onto the surface of the Levator auris longus muscle, GFP-TTC is rapidly concentrated at the neuromuscular junction. Associated intramuscular motor axons were immunostained (red) with an anti-neurofilament 200 antibody.
photo 2: Multiphoton image of a dendritic arbor from a pyramidal neuron within a 400-μ m thick organotypic slice of the CA1 region of the hippocampus transfected with a CAV-Adenovirus expressing GFP-TTC (Scale bar: 15μ m)
photo 3: (A) Ca2+-induced bioluminescence in a cortical neuron expressing GFP-aequorin ,(B) GFP fluorescence ,(C) brightfield images. Color bar = 1 - 9 photons/pixel
photo 4: Calcium wave propagation in Müller cells of the adult mouse retina after ATP stimulation.
A) GFP-aequorin is specifically expressed in Müller cells of the retina after infection with an adenovirus vector, as shown by colocalisation to the Müller cell marker, S-100 β-subunit, but not with the bipolar cell marker, protein kinase C α.
B) Schematic diagram of the retina showing the localization of the different cell types and the corresponding bright field image of an adult mouse retina section.
C) Calcium wave propagation in Müller cells after ATP stimulation
D) Ca2+ responses (photons/s) plotted from six different regions of interest (20 µm diameter). Abbreviations: GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; OS: optic stalk; RPE: pigment epithelium.
photo 5:Cell surface targeted GFP in HEK 293 cells
Keywords: Developmental neurobiology, de-differentiation, synaptic organization, calcium imaging, transgenesis, stem cells