One long standing interest of mine is to understand the mechanisms by which cells interpret and respond to their environment. One such mechanism is direct interaction between neighboring cells. In the context of a developing embryo cell-cell interactions are involved in a variety of processes from tissue morphogenesis to control of organ size and is achieved by a limited number of signaling pathways. The goal of this proposal is to address a fundamental question in developmental biology: How are signaling pathways integrated at the cellular level to give rise to a fully functional organism? Over the last few years, I have been studying Eph receptors and ephrins, evolutionary conserved proteins that provide instructive interactions between neighboring cells. Recently, these studies led me to develop an interest in a different type of cell-cell interaction: direct cell coupling via gap junction communication. Recent studies suggest that Eph receptors and ephrins are potent modulators of stem cell proliferation and apoptosis. The mechanistic details of this regulation remain to be characterized. There is currently tremendous interest in understanding mechanisms regulating maintenance vs. differentiation of NPCs, because of their therapeutic potential. Hence, I chose this system as a model for studying mechanisms of Eph/ephrin signaling in vivo. We will use mouse molecular genetics, cell biology, biochemistry and molecular biology to tackle these questions. The CDA will be invaluable to establish my research group as it will provide both international recognition -which will attract trainees- and financial support to develop my research program.

The complex shaping process of embryogenesis is highly regulated both in time and space at the single-cell level, involving a wide range of control mechanisms. In mammals, most of them are at present unknown or not completely understood due to the lack of reliable in vivo description of cell movements. My project is aimed at imaging whole living mouse embryos in order to map cell movements during gastrulation. I will address the major difficulties that have to be overcome to do this: phototoxicity of imaging, and image quality sufficient for cell segmentation. Phototoxicity will be reduced by performing 2-photon excited fluorescence imaging using an excitation wavelength of 1230nm, which has proven much less destructive for embryos than visible or near-infrared light. I will improve image quality by developing an automated adaptive aberration correction of the excitation wavefront, using an original estimation of wavefront deformation. Finally, I will use these enhanced techniques to study dynamic events during mouse gastrulation, with particular attention to cell movements during the formation of the primitive streak, the three primary germ layers, and elongation of the notochord. This combination of various innovative techniques in optics and embryology should prove powerful to overcome the current limitations of mouse embryo imaging and trigger the development of a complete dynamic atlas of cell fate of highest interest for the comprehension of cell fate regulation in mammals. This interdisciplinary project will provide me new insight both in optics and embryology and will complete my previous research experience in those fields.

Our hypothesis is that the activities of the BDNF TrkB receptor are regulated by recruitment of ubiquitin-interacting proteins that promote internalization, endosomal trafficking and degradation. A recent proteomic approach revealed that among other factors, Hrs and STAM are activated upon BDNF treatment of cortical neurons. Both proteins have established roles in protein sorting and downregulation of the tyrosine kinase receptors EGFR, PDGFR and c-Met, but their importance in neurotrophin receptor trafficking has not been investigated. I will use biochemical and light microscopy techniques to fill this gap and also address at which stage prior to degradation the signal is switched off by monitoring the phosphorylation status of TrkB. In addition, to identify the ligase responsible for TrkB ubiquitination, I will perform an siRNA screen on a human neuroblastoma cell line, using a library that targets ubiquitin ligases. Emerging candidates will be verified by biochemical methods and light microscopy in hippocampal neurons, and their effect on neuronal survival will be tested. BDNF also has an established role in modulating synaptic plasticity. Therefore, in collaboration with Prof. O. Arancio’s laboratory at Columbia University, the relevance of the most promising candidates regulating TrkB/ BDNF degradation will be tested ex vivo by electrophysiological measurements, such as LTP, synaptic fatigue or post-tetanic potentiation, on brain slices. This way we will elucidate if alterations in TrkB and BDNF turnover lead to defects in synaptic transmission. This represents a significant departure from my previous research, which was strictly focussing on cell biology.

IKKalpha, one catalytic subunit of the IkappaB kinase (IKK) complex involved in NF-kappaB activation, is a molecular switch that controls epidermal differentiation. Interestingly, this unexpected function of IKKalpha requires nuclear translocation but not its kinase activity, and is independent from NF-kappaB signaling. IKKalpha–/– mice present a hyperproliferative and undifferentiated epidermis characterized by complete absence of the granular layer and stratum corneum. IKKalpha-deficient keratinocytes do not express terminal differentiation markers and continue to proliferate unless complemented with exogenous IKKalpha. The IKKalpha–/– phenotype is similar to that of IRF6–/– mice, suggesting that IKKalpha and IRF6 regulate common targets in keratinocyte differentiation. Recently, the host laboratory has shown that the Myc family member Mad1, which stimulates cell cycle arrest, is a primary target of IKKalpha in keratinocytes. Upon TGFbeta stimulation, IKKalpha interacts with Smad3 to promote its recruitment to the Mad1 regulatory regions, and to activate Mad1 transcription. To investigate how IKKalpha cooperates with Smad3 to induce Mad1 transcription and whether IRF6 participates in the same process, we will determine if IKKalpha binds directly to a specific DNA sequence in the Mad1 regulatory region and whether it functions to stabilize the formation of a transcriptional complex containing both Smad3 and IRF6.

When we look at our hand we immediately sense it as part of our own body. In this project I wish to address the fundamental question of how the brain produces this feeling of body ownership. My hypothesis is that a key mechanism for body ownership is the integration of information from different sensory modalities (vision, touch and muscle sense) in premotor and parietal cortex. To test this prediction, I will use perceptual illusions that allow me to manipulate the sense of body ownership. I will record brain activity using functional magnetic resonance imaging and electro-encephalography. I will also cause disruption to activity using transcranial magnetic stimulation. A second major goal is to project ownership of a person’s whole body to a “virtual body” by manipulating key processes related to ownership and visual perspective. This will provide fundamental new insights into how we perceive our body in space under normal conditions. I will test the neurophysiological model that premotor and parietal areas signal body ownership and defines the boundary of the body, while medial temporal lobe structures represent the location of the body in the environment. The Career Development Award will allow me to launch a unique interdisciplinary research program on bodily self-perception and virtual bodies, utilizing a wide range of state-of-the-art methods from the fields of cognitive neuroscience, computational neuroscience, and computer science. Furthermore, the CDA will allow me to establish me as the leading international expert on body self-perception, and to form a dynamic research group which will attract top international post-docs.

One of the major goals in neuroscience is to understand how information is represented in the brain. One hypothesis states that information could be contained in the precise spike timing of neurons (temporal coding) notably in the temporal frame of global synchronization of neuronal activity. One of the best examples of temporal coding associated to neuronal oscillations is observed in the hippocampus, a structure involved in spatial memory in rodents and in episodic memory in humans. During exploratory behaviors, principal cells increase their firing in specific locations of the environment (called place fields) but they also fire at progressively earlier phases of the ongoing theta rhythm (4-12Hz oscillations consistently recorded in the hippocampus during exploration) as the animal passes through the place field. The cellular mechanisms of this “theta phase precession” phenomenon remain unknown despite years of intensive extracellular recordings and computational work. In this fellowship I propose to use the newly developed technique of whole-cell recordings in freely moving rats to gain valuable information concerning the cellular events (inhibitory/excitatory drive, changes in intrinsic membrane properties) associated to theta phase precession in vivo in combination with behavioral analysis. I will then use this technique to directly manipulate place cell’s membrane potential through current injections in order to test currently available models of theta phase precession. This postdoctoral project will also give me the unique opportunity to integrate my knowledge on basic mechanisms of synaptic transmission in the new and fascinating field of behavioral neuroscience.

The evolutionary mechanisms by which morphological change is achieved without disrupting function are poorly understood. The diversity of jaw morphology in gnathostomes provides an optimal system to study this question, especially given the high functional demands on these structures. The Depew lab has proposed the ‘hinge and caps’ model to explain how gene expression patterns are elaborated to generate morphologic diversity while maintaining functional competence in jaws. This model predicts the presence of coordinated developmental modules within the axis of each jaw. The proposed project will test the hypothesis that Satb2, a gene recently found to act in a dosage-dependent manner in the regulation of mouse jaw morphogenesis, defines such a module within the hinge and caps system. The goals of this study are to test the hypothesis that the coordinated Satb2 expression observed in mice in the structures that generate the jaws is a shared characteristic among gnathostomes, to evaluate whether Satb2 concentration regulates module morphogenesis in those species in which it is expressed, and to determine how Satb2 expression is induced and maintained. This work incorporates comparative neontology, genomic cloning, and organ and tissue culture systems, which are novel techniques for me. The training I will receive in the Depew lab, together with that from my doctoral work in the cell biology of neurogenesis, will be integral to my future goals of investigating the mechanisms that regulate the coordination of brain and craniofacial development and how these mechanisms have been elaborated during evolution.

The liver is a multifunctional organ with a unique immunological make up that relies on innate immune cells. The most abundant liver lymphocyte population is represented by the innate-like Natural Killer T (NKT) cells, which recognize glycolipid antigens presented by CD1d. Although the liver is a privileged target for viral, bacterial, and protozoan pathogens, the mechanisms of liver specific immunity and the significance of liver NKT cell activation during infection remain poorly investigated areas. I propose to address these questions in the well-established model of murine liver infection by Leishmania donovani that expresses glycolipids known to stimulate liver NKT cells and to participate to the anti-leishmanial host response during infection. This unique experimental system combined with highly inter-disciplinary methods of molecular parasitology, microscopy imaging and glycochemistry will allow us to gain novel insight into mechanisms of hepatic immunology with applicability to other important pathogens, such as malaria, mycobacteria, or trypanosoma, all off which express immunogenic glycolipid antigens. Based on my previous research achievements, and the novelty and significance of this project for infectious immunology, the Pasteur Institute recruited me recently as a project leader. At this early stage of my career, the CDA will constitute a unique financial opportunity, critically needed to develop independently my line of research and to stay competitive at the international level. The award will have a considerable impact for the success of my academic future as a leading scientist in the field of NKT cell-mediated liver immunity.

Communication between cells of the immune system is necessary to control immune responses in time and space, and probably takes an important part in recognition and information processing as well. Cytokines are important mediators of that intercellular communication and have crucial functions in the development, differentiation and regulation of immune cells. The cytokine network is characterized by a large amount of redundancy and pleiotropy, which complicate its understanding. Quantitative studies of basic modules of the network at the single cell level, and under well-controlled stimulation conditions, may enhance understanding of its function. I propose to develop and apply new experimental techniques for quantitative characterization of intercellular cytokine communication between cells of the immune system. The proposed methodology will employ sensitive fluorescent microscopy and ELISA based immunoassay for detection of cytokines secreted by individual cells trapped inside microfluidics chambers. I propose to apply those methods to characterize basic modules involved in T-cell activation and differentiation. Mathematical modeling of network responses, including effects of stochasticity in cytokine expression, will support the experimental results. I believe that the proposed study can advance our ability to decipher the “language” of cytokines, which is essential for our understanding of the immune system and its function as a whole and for our ability to beneficially manipulate it in case of disease.

Precisely regulated synaptic transmission in the brain underlies the molecular basis for learning and memory. The dysfunction of synaptic transmission is implicated in the pathogenesis of various synaptic disorders such as epilepsy. AMPA-typed glutamate receptors (AMPARs) mediate the most prominent form of excitatory neurotransmission in the brain. Dynamic regulation of AMPARs represents a primary mechanism for controlling synaptic strength. We recently identified a novel neuronal ligand/receptor interaction between LGI1 and ADAM22, both of which are genetically related to epilepsy. This interaction specifically enhances AMPAR-mediated synaptic transmission in hippocampal slices. However, how this ligand/receptor interaction regulates AMPARs and why LGI1 and ADAM22 mutations cause epilepsy are still unclear. In the proposed projects, I will study (i) if and how neuronal LGI1 secretion is regulated by synaptic activity (ii) the signaling events downstream of LGI1/ADAM22 interaction and (iii) the physiological and pathological roles of LGI1/ADAM22 in synaptic transmission and epileptogenesis. I will analyze modes of action and regulation of LGI1/ADAM22 interaction by integrating multidisciplinary approaches including living imaging, biochemistry, structural study, and electrophysiology. The detailed understanding of this novel pathway regulating synaptic strength will provide insight into mechanisms underlying learning and memory. The CDA will permit me to build a diverse and productive team including students and technicians and expand international collaborations. Thus, this award will be fundamental to the development of my independent research.

The POKEMON gene (POK-Erythroid-Myeloid-ONtogenic factor, also known as LRF) encodes a novel member of the POK family. POK (POZ and Krüppel) proteins are transcription factors that can bind DNA through a Krüppel-like-DNA binding domain and repress transcription through the POZ (POxvirus and Zinc finger) domain. POK proteins are important in embryonic development, differentiation and oncogenesis. We plan to generate different genetically modified mice to define the role of Pokemon in normal adult life and in cancer pathogenesis. Pokemon inactivation in mice results in embryonic lethality due to a marked impairment in hemopoietic development. We plan to generate mice for conditional inactivation of Pokemon gene in the hemopoietic compartment in an inducible fashion in order to define the role of Pokemon in adult hemopoiesis. Pokemon may offer an effective new target for cancer therapeutics, as recent studies have shown that Pokemon-/- cells are completely refractory to oncogene-induced transformation. By crossing Pokemon conditional knockout mice with lymphoma/leukemia prone strains we will be able to test whether Pokemon inactivation has any tumor protective effect in vivo. Prof. Pandolfi´s group has shown that transgenic mice overexpressing Pokemon in immature T and B lymphoid lineage cells develop thymic lymphomas. We plan to generate transgenic mice overexpressing Pokemon in other tissues to reveal the role of this protein in other tumor types. In addition to these in vivo genetic approaches, we also propose to identify new Pokemon target genes to understand the molecular mechanisms of this transcription factor and how they can be corrupted during tumorigenesis.