The inter-cellular transfer of regulatory transcription factors and mRNAs has become a new paradigm in plant biology in recent years. The transcription factor KNOTTED1 (KN1) was the first such example of a developmental signal that traffics from cell to cell. Its conserved DNA binding domain, the homeodomain, is sufficient to mediate its own transfer from cell to cell in vivo, as well as promoting transfer of the KN1 mRNA. The molecular details and significance of this interaction as well as the significance of KN1 mRNA movement remain elusive. I therefore propose to investigate these questions by: (i) determining the sequences required for the interaction between KN1 and its mRNA, (ii) uncovering the molecular mechanisms of KN1/mRNA intra- and inter-cellular trafficking. (iii) Analyzing the physiological significance of KN1/mRNA trafficking in plant development. This interdisciplinary project aims to study the molecular mechanisms of a new paradigm in intercellular communication: the direct transfer of mRNA and transcription factors from cell to cell. My previous research experience has focused on evolutionary plant molecular biology and genetics. This project will train me in plant cell biology and biochemistry. Finally this project will provide the starting point for me to become an independent investigator.

During nervous system development, neurons have exquisite ability in selecting their synaptic partners among other adjacent cells and forming synapses only onto discrete and stereotyped subcellular regions of these targets. The molecular mechanism of such specificity is largely unknown. In C. elegans a genetic screen for mutants with defects in synaptic specificity has identified SYG-1 and SYG-2, two proteins of the Immunoglobulin superfamily that are both localized at synapses. In both syg-1 and syg-2 mutants, egg-laying motor neuron HSNL fails to recognize its synaptic partners and forms ectopic synapses onto abnormal targets, although its axon trajectory is normal. Interestingly the ectopic synapses in the syg mutants are still formed onto a selective group of targets, suggesting that synaptic specificity is altered but not eliminated in the absence of SYG-1/SYG-2. These data raise the possibility that synapses are formed as a result of synaptic competition between hierarchical potential targets. As a post-doctoral fellow in Dr Shen’s lab I propose to test this model using complementary cellular, molecular and genetic approaches in C. elegans. I will perform time-lapse experiments to ask if synaptic competition occurs during normal development. I will further test whether wild type synapses are required for suppression of ectopic vesicle clustering, by ablation and rescue experiments. Finally I will identify the factors downstream SYG-1/SYG-2 involved in synaptic competition using different genetic screens. In parallel I will develop trans-synaptic markers that will allow functional studies on synaptic specificity and neuronal networks development in C. elegans.

Thrombocytopenia, or low platelet count, is a serious problem in hematological and oncological clinical practice. The best mouse model for this disease is the Mpl-/- mouse, which lacks the receptor for thrombopoietin, and has only 10% of normal platelet levels. Recently, ENU mutagenesis-forward genetic screens have been used in the mouse to investigate gene-phenotype relationships. Thus far, these screens have almost exclusively used wild-type mice as a starting point. In contrast, we plan a large-scale forward genetic screen based on Mpl-/- mice. By screening the offspring of ENU-treated mice for elevated platelet counts we aim to find dominant suppressors of thrombocytopenia. These mutants will be identified and characterised biochemically. Like most small molecule therapeutics, the majority of ENU induced mutations lead to loss of function of proteins. Thus, in addition to the primary aim of significantly advancing our knowledge of platelet biology, these studies may identify new drug targets for the treatment of thrombocytopenia. During my PhD, I have used gene targeting technology or ‘reverse genetics’ to study signalling downstream of insulin/growth factor receptors. While this approach has been invaluable in investigating countless biological systems, it requires prior knowledge of the gene of interest. In contrast, a forward genetic screen selects for mutations in genes that have a function in the biological process of interest. A fellowship will allow me to acquire experience in murine forward genetic screening. I believe this, in combination with the techniques in reverse genetics learned during my PhD, will place me in a powerful position in the future.

The posterior parietal cortex (PPC) has been implicated in sensorimotor integration. Many of its specialized areas have increased firing rates during working memory in delayed-response tasks, but little is known about the neural mechanism that gives rise to the sustained activity. It has been suggested that oscillatory activity in the gamma frequency band (> 30 Hz) could be significant. The current proposal is intended to test this idea in human sensorimotor integration, using magnetoencephalography (MEG). More specifically we will ask whether gamma oscillations play a role in the encoding, maintenance, and updating of spatial working memories for eye and reaching movements. The research will yield significant new results and will have profound consequences for how we think about the computational physiology of sensorimotor control. This CDA makes it possible to develop a new research line, with novel research projects, as described in this proposal. The CDA will enable me to gain expertise with a relatively new neuroimaging technique that would be adequate to ensure future government funding for my research.

Courtship behaviors are highly specialized, innate behaviors essential for propagation in higher animals. Pheromone recognition is the most important sense for this process in many animals, including many insects. Recently, a candidate pheromone receptor with a specific function in male courtship was identified in Drosophila, providing a precedent for a single pheromone receptor gene with a specific behavioral function. Moreover, the gene is closely related to seven additional genes, and it is likely that some or all of them function as additional pheromone receptors. I plan to characterize expression of four members of this pheromone receptor gene family, and I shall determine their specific roles in courtship behavior. At present, little is known about the relationship of individual pheromone receptors and mating behaviors they control. Thus, my studies will establish a novel catalogue of innate behaviors, which are controlled by specific pheromone receptors. Moreover, these investigations will shed light onto the neurobiological and molecular processes of complex behaviors, and they will generate new tools for future studies on their neural circuitry. I have previously studied chloroplast RNA editing, a completely different topic than the one proposed here. However, I have recently become very interested in the general area of neurobiology, with a specific interest in processes that control innate or learned behaviors. Even though this new field is quite different from my previous work as a graduate student, my expertise in molecular techniques is readily applicable to the characterization of pheromone driven processes.

The structural and functional complexity of nerve cells remains one of the biggest mysteries of Neurosciences. How do neurons develop from a cellular sphere into a rich and complex cell? Once developed, how are neurons maintained in the highly dynamic and changing environment of the brain? These questions lie at the heart of brain research and, yet, remain poorly understood. Here, we propose to combine two-photon microscopy and lentivirus gene technology to develop an experimental model for studying dendritic development and synaptogenesis, in vivo. Specifically, we will study a population of neurons in the adult brain that continues to develop well into adulthood. This unique neuronal population is a pool of progenitor cells lining the lateral ventricle, which migrate into the olfactory bulb and differentiate into functional inhibitory neurons of the local circuitry. Using lentivirus transduction we will genetically tag and manipulate the newborn neurons. Using in vivo imaging we will study these neurons as they develop and integrate into functional networks. These experiments will provide new insights into the mechanism of dendritic and synaptic development as well synaptic maintenance in the intact mammalian brain. The CDA will enable our lab to acquire the necessary independence with regard to the genetic manipulations of this experimental system. Since synaptic development is a fundamental topic in the Neuroscience field, other areas of research such as physiology, molecular biology and behavior are necessary for a comprehensive understanding of this system. Thus, the CDA will prompt collaborations of my lab with other researchers in other fields and countries.

The aim of my proposal is to understand the fundamental role of chromatin modifications on RNA Polymerase II (RNAPII) regulation. To do so, I will use the retrotransposon Ty1 in S. cerevisiae as a biological model. The promoter of Ty1 is incredibly complicated with many features characteristic of higher eukaryotes and understanding its regulation provides fundamental information on transcription control. Among these aspects, Ty1 is subject to a unique transcriptional silencing by cosuppression mechanism. Transcriptional cosuppression involves RNA interference, histone or DNA methylation. Interestingly, S. cerevisiae exhibits none of characterized features identified in other organisms and Ty1 cosuppression may represent a novel regulation involving RNAPII, RNA maturation and chromatin remodeling. To understand this Ty1 specific transcription control, I will use 2 major approaches. Firstly, I will characterize Ty1 transcriptional dormancy using a range of chromatin techniques (Chromatin Immunoprecipitation coupled with real-time PCR analysis, Transcriptional Run-On, Chromosome Conformation Capture and chromatin analysis). Secondly, I will identify genetically factors affecting Ty1 transcriptional cosuppression. The use of recent and accurate techniques to detect chromatin changes and RNAPII dynamic is very demanding in financial supports. HFSP CDA will dramatically help me to set up an appropriate and competitive laboratory environment to develop this project and organize a collaboration network through laboratories with different and complementary expertise. Finally, this Award will enable me to begin building a team which is essential both technically and intellectually.

The present proposal focus on the role of the BTAF1 protein in transcription regulation. DNA promoter recognition and subsequent assembly of transcription complexes depend on TBP (TATA-binding protein) containing complexes. Among them B-TFIID is unique in containing only one TBP interacting protein, BTAF1 and in exhibiting ATPase activity which regulates BTAF1-TBP interaction. Little is known about the regulation of B-TFIID/BTAF1 activity. The proposal aims i) to identify new interactors of BTAF1 and to study effects of them on BTAF1 function both in transcription and in ATPase assays and ii) to visualize conformational changes within B-TFIID. To understand more precisely the chromatin remodelling function of BTAF1 in this process, emphasis will be made on the Myb partners of BTAF1, transcriptional activators involved in some cancers. The laboratory of Marc Timmers will act as my host and is located at the University Medical Centre of Utrecht in the Netherlands. This laboratory is recognized as expert concerning the regulation of transcription. With my background in structural biology and more specifically in NMR spectroscopy of membrane and soluble proteins acquired up to the end of my Ph.D., this project essentially based on cellular biology, biochemistry of DNA-protein interactions and electron microscopy represents a new promising challenge and will be useful for carrying on my research career.

The phylogenetic position of Xenoturbella has long remained obscure, and although they were recently reported as deuterostomes, further studies are essential to confirm their phylogenetic position. By revealing their life cycle and gathering the traits showing the evolutionary history of the animal, I aim to elucidate the phylogenetic position of the animal. If they are confirmed to be deuterostomes, new knowledge of deuterostome evolution will be obtained through this project. The early history of deuterostomes, to which we belong, remains unclear, but Xenoturbella may represent the simple body plan of the last common ancestor of the deuterostomes. Research on their morphology, gene expression patterns, and embryology should reveal important features of the early evolution of deuterostomes and the body plan of their common ancestor. The first important task will be examining the reproductive cycle of Xenoturbella. The breeding season will be determined followed by induction of spawning and/or embryo release. Developmental stages will be reared to adults. Examples of each stage will be fixed and analyzed by several morphological methods. In addition, expression patterns of developmental regulatory genes will be investigated to better understand the structure of Xenoturbella. Molecular studies will be a new experience to me. As molecular techniques become essential to most areas of biology, it is vital for me to acquire them early in my career. I will also learn ecological methods and establish a long-term culture system for Xenoturbella. Long-term culture of live specimens will enable observation of natural spawning, mating behavior, fission, and regeneration.

The entorhinal cortex (EC) acts as a gateway for information flow to and from the hippocampus and neocortex, and plays a crucial role in various memory-dependent tasks. It has been postulated that principal neurons in layer II (LII) of the EC mediate a comparison of hippocampal input and output signals, making the EC part of a novelty detection network. While hippocampal inputs reaching LII EC neurons are from the neocortex, hippocampal outputs reach them through intra-entorhinal projections from layer V. The objectives of this proposal are to test if principal neurons in LII act as novelty detectors and to study the ionic mechanisms underlying such function. I propose to achieve these goals by using a combination of experimental and theoretical techniques. First, I plan to use electrophysiological and imaging techniques to experimentally characterize dendritic information processing. Specifically, I propose to (i) study spike timing-dependent plasticity (STDP) of intra-entorhinal and cortical projections onto LII EC neurons (ii) assess the roles of dendritic ion channels in novelty detection by presenting LII EC neurons with paired inputs from these two pathways and using blockers for various ion channels and (iii) analyze the possibility that STDP of either pathway also causes plasticity of dendritic ion channels. Then, I will build morphologically realistic models of LII EC neurons taking into account their intrinsic properties and the outcomes of the above experiments. Finally, feeding these model cells with paired inputs with varied synaptic strengths and timing, I plan to test the relevance of these experimental findings to novelty detection at the systems level.

The cytoskeleton is the physical backbone that maintains the architecture and integrity of eurkaryotic cells and especially, of CNS neurons, featured by complex dendritic arborization and long axon that can reach one meter in Human. In early life, a destabilization of this highly ordered dynamic structure triggers neurodevelopmental disorders while in the adult CNS, cytoskeletal alterations promote neurodegenerative disorders. The interconnection of three networks- microfilaments (MFs), intermediate filaments (IFs) and microtubules (MTs)- builds the cytoskeleton. Enriched in CNS neurons, MTs serve as tracks for intracellular transport through molecular motors to deliver neurotrophic factors, organelles, and vesicles to appropriate cellular compartments. With MFs and IFs, MTs modulate the shape of the neuron in accordance to its developmental stages, maturation, energetic needs and functions. Thus, regulation of MTs dynamics and stability by Microtubule associated proteins (MAPs) impacts directly on the behavior of the neuron. Protein 600 (p600) encodes for a polypeptide of 5184 amino acids (GenBank Accession Number: NP_065816) with unknown functions in the CNS. The objective of the present research project is to define the roles of p600 in the CNS. Based on its homology with proteins linked to cytoskeleton-mediated transport, p600 may constitute a novel cytoskeleton-associated protein and, specifically, a novel MAP that couples the development of the CNS to its maturation and demise. Along with my interest for studying the cytoskeleton, this project will eventually permit me to build career and develop independent research.

The striatum is a brain region involved in motor coordination and cognitive function. Several degenerative disorders are associated with striatal dysfunction, including Parkinson’s and early stage Alzheimer’s diseases. Symptoms appear to be caused by an imbalance of synaptic transmission, in particular of the slow modulatory synaptic signals provided by dopamine (DA) and acetylcholine (ACh) to the main striatal cell type, the medium spiny neuron (MSN). The effect of DA and ACh on synaptic processing in the striatum is unclear. The proposed research addresses the controversial question of whether DA and ACh regulate postsynaptic channels in MSNs, specifically: a) do DA and ACh regulate postsynaptic glutamate receptors? b) do DA and ACh regulate the calcium influx evoked in spines by action potentials in MSNs? and c) what is the amplitude, timecourse and spatial extent of modulation following synaptically released neuromodulator? Post-synaptic channels directly in the spine will be examined using optical methods that allow rapid local application of neurotransmitter, thus avoiding confounds from DA and ACh modulation of glutamate release. Optical methods also permit the identification of modulatory synapses in cell pairs, which would otherwise be difficult because these synapses are not associated with the fast currents usually used to indicate the presence of a connection. This project would add experience with optical experiments to my skills in electrophysiology and mathematical modelling. In addition, I would like to extend my training in synaptic biophysics to more complex processes including synaptic integration and neuromodulation.

Growth and development of a plant requires precise control and co-ordination mechanisms of carbon assimilation, transport and storage. In spite of the fundamental importance of these processes –almost all of the organic carbon on earth is derived from them– crucial aspects remain poorly understood. Progress in understanding carbon metabolism, transport and signalling is severely hampered by a lack of knowledge of sugar concentrations inside living cells in intact plants. The proposed project aims to overcome these obstacles: During the first phase of the fellowship I will design and create a sucrose nanosensor and use it together with existing nanosensors to study glucose, maltose and sucrose levels in living cells under different environmental and genetic conditions. In the second phase I will apply these techniques in combination with functional genomics techniques to study mechanisms controlling carbon partitioning. This approach will lead to deeper understanding of how carbon availability controls growth and development of the whole plant. The proposed research differs from my previous experience in its interdisciplinary nature. I will be using latest techniques in photometrics, microscopy, computational design of proteins, computer modelling and functional genomics to answer key problems in biology. I will be supervised by an expert in transport physiology and nutrient sensing and collaborate with a photometrics specialist to ensure that I will acquire expertise in both fields. Most importantly the project is expected to generate information that will provide the basis of major new research programs, on which an independent research career can be based.