The superfamily of TGF-ß ligands represents one of the most prominent families of morphogens. These factors have profound effects on many aspects of embryonic development, cell behaviour and homeostasis and malfunction of the pathways associated with these cytokines can lead to a variety of pathologies. Despite intensive research, there are still large gaps in our knowledge regarding the specificity of these ligands, the regulation of the activity of their receptors and the interactions between the TGF-ß pathways and other signalling pathways. In particular, how sources of TGF-ß morphogens are generated and how the resulting morphogen gradients can be translated into patterns of gene expression during early development remain central questions in current developmental biology. This proposal attempts to fill these gaps in our knowledge by characterising novel regulators of dorsal-ventral axis formation upstream and downstream of Nodal and by modelling the gene regulatory network (GRN) activated by this factor. We address this question within the sea urchin model, an organism phylogenetically close to vertebrates but with many advantages for the analysis of regulatory networks in early development. Our first aim is to characterize the early events that shape the Nodal gradient and initiate the downstream GRN that controls dorsal-ventral (D/V) axis formation. Our laboratory has recently discovered several key factors involved in D/V axis formation. We identified a maternal TGF-ß ligand, a transmembrane protein, an ETS domain transcriptional repressor and the JNK kinase as factors critically required to restrict the spatial expression of nodal. The similarity of the phenotypes caused by inactivation of either this maternal TGF-ß ligand, the transmembrane protein or this ETS factor strongly suggests that they act in the same pathway to specify the D/V axis. However, the relationships between these factors and the mechanisms by which they antagonize nodal expression are presently completely enigmatic. To clarify the relationships between these factors we will identify the binding partners of the TGF-ß ligand and perform biochemical analyses and epistasis experiments. In this first aim, we will also determine whether Nodal and/or BMP2/4 work as morphogens, i.e. as long-range, concentration-dependent signalling factors in the sea urchin embryo by using a combination of treatments with recombinant Nodal and BMP2/4 proteins and ectopic expression of mRNAs encoding these ligands or the activated forms of their receptors. The second aim of this project is to extend and model the gene regulatory network activated by Nodal. We recently identified and validated about fifteen novel genes regulated by Nodal encoding a variety of regulatory proteins including transcription factors, cytokines, and secreted proteins most of which have never been characterized. The expression patterns of these genes identify novel regulatory domains and boundaries along both the animal-vegetal and dorsal-ventral axes, revealing an unsuspected complexity in patterning of the ectoderm. We propose to dissect the regulatory mechanisms establishing these new domains, to characterize these novel Nodal target genes and to analyze their function and position in the GRN. Finally, to further test the role of individual components of this network and understand how it achieves both robustness to environmental perturbations such as regulative development, and plasticity to evolutionary scenarios, we will start to construct a logical model of this GRN. Our third and last aim is to start dissecting the mechanisms that allow responding cells to read different levels of Nodal or BMP2/4. We will start to investigate how thresholds of response are encoded in the genome. We will perform detailed bioinformatics analyses on an extended set of Nodal and BMP2/4 target genes to identify and dissect the architecture of the cis-regulatory modules of these selected target genes.

In the past few years, we have developed optical, imaging and analytical tools to monitor the behavior of neurotransmitter receptors at the single molecule level. This allowed us to access novel dimensions of neurotransmission regulation. Based on this novel understanding of synaptic biology, we have demonstrated that glycine receptor (Gly-R) and GABAA-R lateral diffusion and capture at synapses are regulated by activity. We also showed that the coupling excitation-inhibition is regulated by calcium dependant phosphorylation. Based on these tools and the new understanding of synapse, we now propose a new project to access novel dimensions of the physiological non-cell autonomous regulation of inhibitory neurotransmitter dynamic and synaptic strength. It will also decipher their pharmacological controls. Task 1: we found that the diffusion dynamic of Gly-Rs is regulated via a PKC-dependent mechanism. We identified the S403 in the intracellular loop of the Gly-R as the target of PKC. We will now unravel the regulation of GABAAR diffusion capture. We will then establish how these regulations are used to fine-tune the strength of synaptic inhibition upon neuronal activity. Task 2: our preliminary data establish that microglial activation down regulates Gly-R and GABAA-R trapping at synapses leading to a disinhibition. We propose to determine how microglia and astrocytes are involved in this disinhibition. We will then focus on the role of TNFa and Thrombospondin that are glial factors for which we have preliminary results. This will lead us to describe how the homeostatic balance between excitation and inhibition is (dys)-regulated upon microglial activation. Task 3: we have evidence that the metabotropic GABAB receptors (mGABAB-Rs) modulate Gly-R and GABAA-R lateral diffusion and interactions with scaffolding proteins, ultimately controlling their accumulation at synapses. We will determine how the trafficking of Gly-R and GABAA-R are impacted by GABAB signaling, and unravel the mechanisms of this regulation. This will provide a basis for pharmacological actions targeting inhibition. Finally (Task 4), we have developed a multicolor super-resolutive PALM-STORM microscope (resolution about 20nm) to access novel dimensions of the dynamic of membrane proteins. We will combine the super-resolution with Single Particle Tracking. Our aim is to collect quantitative molecular data together with dynamic aspects of receptor binding to allow a biochemical study in cellulo. Ultimately, we will put a strong effort to access super-resolution imaging of Gly-R and GABAAR diffusion in integrated systems such as organotypic slice cultures. The tools developed in this part of the project will be used in all the aspects of the project related to receptor dynamic. Ultimately this research may find potential applications in situations were the homeostatic excitation/inhibition regulation is altered following inflammatory microglial activation resulting in increased glutamatergic neurotransmission or loss of tonic inhibitory control as seen in sensitization of pain or in other synaptopathies such as spasticity and epilepsy. Selected publications of the partners as corresponding authors: Pascual et al (2011) PNAS ; Pinaud & Dahan (2011) PNAS; Specht et al (2011) EMBO J; Charrier et al. (2010). Nature Neurosci; Renner et al. (2010) Neuron; Pinaud et al. (2010) Nature Meth; Bernard et al. (2010) EMBO J; Bannai et al. (2009) Neuron; Lévi et al. (2008) Neuron; Triller & Choquet (2008) Neuron; Bouzigues et al (2007) PNAS; Dahan et al.(2003) Science 302:442.; Meier et al. (2001) Nature Neurosci 4:253

Gene expression is a dynamic process involving a cascade of events happening at different size and time scales, from macromolecules to chromosomes (nm to µm), and from the diffusion of small molecules to the transcription of megabase long genes (µs to h). Using fluorescent tags, it is possible to: a) check for the presence of molecular complexes in live cells, using FRET, b) investigate the mobility of such complexes, using photobleaching methods such as FRAP and FLIP, Correlation Spectroscopies (FCS, FLCS, FCCS, ICS) and Single Protein Tracking (SPT). These different techniques cover different time scales and each have their own limitations. Photobleaching methods require high concentrations of labelled proteins, temporal resolution of FRET is limited by acquisition times (from seconds to minutes), and FCCS is limited to time ranges between 1 microsecond and 1 second. SPT is not easily amenable to high throughput. Therefore, using only one of the above-mentioned techniques in isolation is not enough to obtain robust, interpretable results and an integrated approach is required. Studying positive transcription elongation factor (P-TEFb) provides an attractive model of how individual molecular events are coordinated and regulated during a process as tightly regulated as gene transcription. This factor is essential both for productive transcription by RNA polymerase II, and for transcript processing. Using biochemical methods, the Bensaude lab and others have already established that P-TEFb mainly exists as part of a mixture of two populations of complexes markedly differing in size, subunit composition and activity. The “small” (?4.5 nm Stokes radius) core P-TEFb complex is active, but becomes inactive when it forms the “larger” (?11.6 nm Stokes radius) complex with the HEXIM1 protein and a core 7SK small nuclear ribonucleoparticle (7SK snRNP). In response to physiological stimulations, or when transcription is arrested, the larger P-TEFb complex dissociates. Interestingly, preliminary data obtained by this consortium, using FRAP, FLIP, FCS and SPT techniques independently, unexpectedly suggested that the larger complex might be more mobile than the smaller one. This result opens new perspectives on the possible roles of HEXIM1/7SK binding, by suggesting that the “inactive” P-TEFb complex might reach its target faster than the “active” one. Since preliminary results also suggested that the mobility of the P-TEFb complex was regulated, potentially via a chaperoning/carrier effect, we here propose to focus on how this regulation may occur. To do this, we will investigate the mobility of the P-TEFb complex in relation to its composition. The contributions of different molecular interactions will be dissected using a multi-approach imaging strategy. This will enable us to determine: 1) which interactions interfere with P-TEFb mobility; 2) the half-life of the P-TEFb/HEXIM1/7SK complex; 3) whether core and large P-TEFb complexes have differing access to the nuclear space; 4) whether HEXIM1/7SK binding affects the recruitment/release of the P-TEFb complex to active genes. One of the major limits of our preliminary work was the poor definition of the macromolecular species we observed. To overcome this problem, we will use genetic approaches. We will generate P-TEFb mutants, which cannot assemble, or assemble more stably than wild-type with HEXIM1/7SK. The behaviour of these mutants will be investigated in live cells, using a combination of Photobleaching, Correlation Spectroscopies and Single Particle Tracking methods. Furthermore, a bimodal microscope coupling FCCS and FLIM (FLCS) will be developed to determine the diffusion characteristics of the identified complexes. The combination of genetic tools and state-of-the art imaging technologies is a novel approach, which will enable us to study biochemical reactions at the molecular level in live cells, and should reveal important clues about the function and regulation P-TEFb.

In this project we propose to explore, using a multilevel approach, the role of unconventional NMDA receptors (NMDARs) in specific regions of the adult brain involved in higher cognitive functions. Much is indeed known about the central role of the family of conventional GluN1/GluN2 NMDARs in glutamatergic transmission during brain function and dysfunction. In contrast, there exists a largely underappreciated group of glycine-binding NMDAR subunits, GluN3A and GluN3B, which have been so far uniquely associated with synaptic maturation and plasticity at early developmental stages. Our project lies its foundations upon exciting preliminary results showing that there are specific regions of the adult brain- the epithalamus (medial habenula, MHb) and the thalamus (intralaminar and midline nuclei, IL/MDL) - where GluN3A is expressed at high levels, and where it participates to several subtypes of NMDARs including a completely novel type of neuronal excitatory glycine-activated receptors. The MHb participates to the expression of a large variety of aversive physiological states, whereas the IL/MDL nuclei are likely at the heart of the generation and the maintenance of attentional vigil states. The intimate association of GluN3A subunits with these brain territories raises important questions about the rules and roles of GluN3A receptors in adult brain function. By unveiling a novel receptor target, it also bears potential from a therapeutic perspective. We will investigate how and when GluN3A receptors are activated and what is their impact on local circuit function and on higher cognitive functions linked to behavioral tasks. To do so, we will combine a broad spectrum of different technical approaches ranging from ion channel sequence engineering to optogenetic-based mapping, in-vitro electrophysiology and behavioral tests associated with EEG and optical recordings in freely moving animals. In particular, we will use available genetic tools (KO, shRNA, genetically-modified mice) and innovative molecular tools including GluN3A-containing receptors whose function can be reversibly and specifically manipulated by light. These instruments will allow us to achieve a molecular level control of GluN3A-NMDARs that is subunit-specific, reversible within a time scale compatible with fast neuronal communication, and usable both in vitro and in vivo in behaving animals. We aim at providing key insights of GluN3A function at the biophysical, cellular and behavioral level. At the cross roads of neuroscience and protein engineering, this multi-scale project aims at providing key insights into an underexplored class of receptors with important implications both for adult brain physiology and pathology. Furthermore by implementing original light-controllable receptor tools, our project should boost the emerging field of molecular optopharmacology in vivo. This project involves four internationally-recognized neuroscience groups, including three French groups (Diana, Paoletti, Dieudonné) and one Canadian lab (Kieffer) with a solid experience in collaborative networks. The synergism between the involved teams creates a strong research environment for successful implementation of GluBrain3A. At the outcome of the GluBrain3A project, we envisage unmasking the functional importance of a neglected receptor class and unveiling the functional benefit of the association of this receptor family with specific forebrain relay nuclei critically involved in the regulation of motivational states.

The chemical synapse plays a central role in communication between neurons. Ligand-gated ion channels responsible for synaptic transmission were discovered more than four decades ago and have attracted much attention in neuroscience. Devastating neurological diseases arise when these protein receptors go awry. Most of the prior research has focused on understanding molecular architecture, conformational changes, interactions, and trafficking of individual receptor molecules separately. There has been a recent trend in investigating complex neuronal activities at the systems level. Since receptors are considered to be the “switches” of the neuronal networks, it is essential to understand them under a system context with unified perspective. The unification proposed in this project is anchored on a recent biotechnological innovation in which I (the main coordinator) possess an expertise: the unnatural amino acid (UAA) site-directed mutagenesis. The project will focus on two critical aspects of the technology : 1) to demonstrate the feasibility to incorporate various UAAs in two types of ligand-gated ion-channels and 2) to identify new structural and dynamic properties of the receptors. The development will bring together experts from chemistry, electrophysiology, and high-resolution fluorescent imaging. The combination of several techniques involved will help understand the mechanisms of receptor functions from multiple aspects. We have designed the project into a series of well-defined tasks which focus on the incorporation of two kinds of UAAs in ligand gated ion channels: UAAs that are photoreactive ; and UAAs that serve as chemical handles that can be conjugated with spectroscopic probes. We aim at 1) identifying key structural elements regulating channel activities through a novel photocrosslinking approach; 2) engineering ligand gated ion channels that can be activated by light stimuli; and 3) imaging receptors in their native environment using high resolution fluorescent imaging techniques (stochastic optical reconstruction microscopy and fluorescence correlation spectroscopy) to understand trafficking and diffusion properties. The establishment of those approaches may not only lead to discoveries, but also can be translated to studies of many other receptors. In addition, the combination of these approaches in the studies of a specific receptor will facilitate the emergence of coherent understanding of receptors and their physiological functions. To further demonstrate the power of the UAA mutagensis technology, we will integrated a task to be conducted with an external collaborator in the University Paris-Sud 11 (Institut de Génétique et Microbiologie) centered on the identification of novel ribozymes (catalytic RNAs). The emerging team will perform key functions in the field of chemical biology: advancing the UAA site-directed mutagenesis by developing novel methods to address questions related to ligand-gated ion channels and synaptic transmissions; therefore, importing important technology into the field of neuroscience. By collaborating with experts in fields other than neuroscience, the project will help promote the application of the technology in a variety of problems.