Ascidians are invertebrates with a basic chordate body plan. The extent to which the ascidian larval central nervous system (CNS) diverged from the complex vertebrate CNS provides information about key innovations during chordate CNS evolution. Gene expression patterns of members of the Hedgehog (Hh) signaling pathway in ascidians suggest conservation of signaling pathways involved in chordate CNS development. The application of technologies now available in ascidian research permits functional studies and the characterization of regulatory networks underlying formation of the CNS. Here we would like to focus on Shaggy/GSK3ß, which acts at the interface of the Hh and wingless/Wnt signaling pathway in both Drosophila and vertebrates. Analysis of Shaggy/GSK3ß function in the ascidian Ciona intestinalis will provide insight in the evolution of signaling pathways important for CNS development. Research on these signaling pathways in ascidians also offers a significant advantage over mouse research transgenic animals can be produced much faster, and the genome of ascidians is far less complex. In addition, the host laboratory is the only institute in Europe that performs genetic screens with ascidians. This presents the unique opportunity to screen for ascidian mutant phenotypes on a single cell level, which is just not possible in any vertebrate system. The host laboratory also has a long-standing interest in ascidian research and will provide an excellent environment to study ascidian biology. In combination with the research opportunities and recent advances in this field, this fellowship creates the possibility to become an independent researcher in a new promising field.

Although cell membranes are important sites of biological regulation, function of membrane proteins is poorly understood. Intramembrane-cleaving proteases (I-CliPs) are a recently-discovered class of enzymes involved in regulating membrane protein activity. I-CliPs are novel polytopic membrane proteins that catalyse proteolysis in the hydrophobic membrane bilayer, unlike all other hydrolytic enzymes. Despite their central roles in regulating fundamental biological processes in all branches of life, little is known about how I-CliPs function mechanistically, and about their full roles in different species. I propose to investigate how these novel proteases function in the membrane bilayer using chemical inhibitors that I will develop for this purpose. In a complementary approach, I will probe their function using biochemical and biophysical methods. Finally, I will use these inhibitors to analyze the biological function of one large family of I-CliPs whose members are conserved in all branches of life. My doctoral research was focussed on investigating the roles of cell signalling in embryonic development using the Drosophila model system. Through a combination of genetics and cell biology, I discovered that a key developmental regulator encodes an I-CliP. Unlike my previous experience where I used a genetic and cell biological model to understand cell signalling and development, I now propose to study the biochemical function of this unusual class of enzymes using chemical and physical methods. I have only limited experience in these approaches, and acquiring these skills will be essential to my long-term goals of investigating membrane proteins involved in cell signalling.

Asymmetric distribution of mRNA is an intrinsic mechanism targeting proteins to their site of function. Axis formation in Drosophila requires the localisation of a transcript encoding a TGF alpha homologue, gurken (grk), first to the posterior of the oocyte and later to the dorsal anterior corner. The mechanism of grk mRNA localisation is poorly understood because of the lack of in vivo assays for localisation and the complexity of the process. My project is based on a new in vivo assay, which consists of injecting fluorescently labelled grk RNA in the oocyte and visualising its movement in particles in real time. My goal is to characterise the minimal cis-acting sequences necessary for grk mRNA particles assembly, movement and anchoring. To achieve this, I will inject truncated grk RNAs and study their movements in the oocyte. In parallel, I will develop a new technique, which we call in vivo-SELEX, to define short RNA sequences localising in the oocyte. Another objective is to determine trans-acting factors specific for the different steps of grk movement. I will first investigate the role of known proteins whose mutations induce grk mislocalisation. Moreover, the grk RNA localisation signals will be used to isolate specific binding proteins from oocyte extracts. The candidates will be analysed by mass spectrometry and their role investigated in vivo using mutants or antibodies. This work will allow me to expand my knowledge in cellular biology and biochemistry. The use of powerful imaging systems will complete my knowledge of fluorescent microscopy and of the different acquisition systems, which is essential for my future research carrier.

Cyclin E is one of the activators of Cdk2, expressed near the G1-S phase transition and critical for initiation of DNA replication and other S-phase functions. Protein levels of cyclin E peak at the G1-S boundary, achieved by periodic transcription followed by auto-phosphorylation dependent proteolysis. Deregulation and elevated levels of cyclin E are associated with malignancy possibly through genomic instability. Recently is has been shown that degradation of phosphorylated cyclin E depends on a SCF-complex containing the newly identified human F-box protein hCdc4. hCdc4 is homologous to the yeast Cdc4p and contains WD40 repeats involved in substrate recognition. Both in humans and in mice, three splice variants of Cdc4 are expressed. Biochemical evidence from S. cereviciae and S .pombe indicates that the hCdc4 homologues need to form homo- or heterodimers respectively in order to function properly. Here it is proposed to test dimerization between the different splice variants of hCdc4. Experiments have been designed to reveal the role of the hCdc4 dimerization in relation to substrate specificity, activity of the SCFhCdc4 complex and the possible autoregulation of hCdc4 levels. The proposed project will focus on the role of Cdc4 dimerization, both in lower eukaryotes and in higher eukaryotic systems. Therefore, this project will extend my training to gain expertise with different model systems and further improve my knowledge of standard and novel biological techniques with a particular focus on the biochemistry and cell biology of mammalian tissue culture systems.

Heterochromatin is a specialized chromatin structure present in all eucaryotes with essential functions in chromosome segregation, maintenance of genome stability and transcriptional gene silencing. Recently, a surprising link between heterochromatin formation and the RNA interference (RNAi) pathway was discovered in the fission yeast Schizosaccharomyces pombe. RNAi is a widespread silencing mechanism mostly known for its post-transcriptional activity that degrades target mRNAs or blocks their translation, but studies in plants, Drosophila, Tetrhymena and S.pombe clearly demonstrated that RNAi also acts at the chromatin level. The aim of this proposal is to identify and dissect mechanisms through which RNAi acts at the level of chromatin in S.pombe. Using a protein purification strategy, we have recently purified a complex, that we named RITS (RNA-induced Initiation of Transcriptional gene Silencing)(1). The identification of RITS provides a direct physical link between RNAi and heterochromatin formation machineries and our first studies suggest that RITS complex targets specific genomic regions through an RNA-based pairing mechanism. We are now focusing on understanding how this targeting mechanism works and how it is regulated, using in vitro and in vivo approaches. 1. A. Verdel et al., Science 303, 672-6 (Jan 30, 2004).

The Arabidopsis root has a simple radial structure, organised in concentric single layers. This radial pattern is elaborated during embryogenesis and maintained postembryonically through asymmetric cell divisions of initials in the root apical meristem. The SHORT-ROOT (SHR) gene, encoding a putative transcription factor, is required for the asymmetric cell division of the cortex/endodermis initials, and for endodermis specification. Recent results have revealed that the functions of SHR in root radial patterning depend on intercellular movement of the SHR protein. The aim of this proposal is to find new components of the SHR radial patterning pathway. A genetic screen will be performed to identify mutations that perturb the spatial distribution of the SHR protein. These might affect genes encoding regulators of either SHR expression or SHR protein trafficking. The most interesting mutants will be genetically and phenotypically characterised to determine the role of the mutated genes. One or two mutants will be chosen for map-based molecular cloning. In parallel, conditional expression of SHR coupled to microarray technology will be used to find direct targets of SHR. The function of the putative targets will be analysed by characterising the effect of their disruption or misexpression. During my previous research experience, I become interested in the mechanisms controlling plant development. So far my work was essentially focused on genetic and cytological approaches. This proposal would allow me to look at plant development from a more molecular and genomics point of view. I would acquire tools to identify and to study new genes involved in a developmental pathway.

Brassinosteroid (BR) is multifunctional growth regulator in plants.The aim of this project is to identify new components in the BR signaling cascade, using two different approaches, genetic and genomic, that are based on specific tools available in the host laboratory. Classical genetic based on loss-of-function mutation is not suitable in this particular case due to genetic redundancy, or alternatively to the fact that signaling components may be encoded by essential genes. Genetic analyses will therefore be carried out to isolate gain-of-function mutation, by activation tagging, conferring resistant to BR biosynthesis inhibitor brassinazole. This overexpression of genes at random in the genome is a convenient way to identify components involved in the signaling cascade of physiological events. In parallel, a genomic approach using the Affymetrix technology will be developed to identify genes involved in the signaling pathway and in the early response to BR treatment. The skills required for this project are totally new for me as my PhD and my former experience were mainly characterization of genes involved in a cellular response. This project would allow me to acquire expertise in the field of signal transduction and genomic. Moreover, this project will require bioinformatics for the analyses of genomic data. The skills required can serve to any topic in the field of molecular biology. I have the opportunity, after this postdoctoral position, to come back in the team where I did my PhD as a researcher. I therefore would like to design genetic screen to unravel the signaling cascade leading to the activation of iron transporters in roots described in the achievements.

This project aims to investigate the interactions between the cytoskeleton and trafficking of MHC class I/II molecules in DCs during maturation and contact with T cells in vivo by real-time confocal imaging of MCMV-infected cells. This will be achieved by combining the expertise of the host lab in the area of cell biology and the newly established MCMV-BAC-based mutagenesis methods invented by the applicant. DCs capture antigens in the periphery, migrate to lymphoid organs and present processed antigens via MHC complexes to T cells. Recent in vitro data indicates that during all these processes major changes in the cytoskeletal network of DCs are important(1,2,3). Since in vitro culture systems do not adequately reflect the situation during these processes as they occure in live cells, the project will investigate DCs both in vitro and in the context of the skin and lymphoid organs of the live mouse. We aim to achieve this by using recombinant MCMVs as expression vectors for fusions of GFP (or yFP, cFP) with MHC class I/II, actin, fibronectin, fascin and tubulin. Engineering of MCMV can be easily performed using BAC technology. Since several markers will be co-expressed from one MCMV vector and since MCMV infects various cell types including DCs in mice, this project will for the first time allow us to monitor intracellular interactions in vivo, without laborious generation of transgenic mice. My current research work concentrates on genetics of herpesviruses and on investigation of virus-host cell interactions. In contrast, the proposed project intends to exploit recombinant MCMV as an expression vector for real-time investigation of DCs both in vitro and in vivo.

The subject of the proposed research is the mechanism of mitochondrial fusion. Several factors have been implicated in this process, but their biochemical function and the mechanism of the reaction are not yet known. Therefore, I propose to characterise the biochemical activity of yeast fzo1p, a key player in this process and to purify interaction partners by affinity chromatography. Interacting proteins will be tested for a function in mitochondrial dynamics in vivo. To gain insight into the mechanism of mitochondrial fusion, I will establish a content mixing assay. With such a quantitative assay, the individual steps of the reaction and their different requirements will be determined. I will use yeast as a source material for the assay, because it allows combination of biochemistry with genetic methods. This approach has been extremely powerful in the dissection of the secretory pathway. The proposed research differs from my previous work in the aim of the project as well as the model organism used. However, it will draw on my experience with biochemical assays and similarly to my previous work, relies on the possibility to elucidate cell biological processes by reducing them to an in vitro reaction. This will be augmented by the possibility to validate findings in vivo using yeast genetics. I wish to later expand this research to the regulation of mitochondrial dynamics and its coordination with other organelles. This network can only be understood if one can know the quantities of all factors, which is at the moment only feasible in yeast. The proposed research would make this possible by introducing me to this model system and by expanding my biochemical knowledge.

1) c-Myc is a key regulator of proliferation that is frequently deregulated in tumours. Paradoxically, c-Myc also induces apoptosis. Mitochondria are the principal target for the apoptotic transcriptional action of c-Myc. To identify c-Myc effectors a proteomic approach will be employed to determine Myc-induced changes in mitochondrial polypeptides. These proteins will be identified and their roles in Myc-induced apoptosis assessed. 2) The project differs from my previous research in that I will study dividing cells and learn about the cell cycle, with which I am unfamiliar (my research so far has involved post-mitotic neurons). The focus on the c-Myc oncogene and cancer contrasts with my current work studying the excessive cell deaths that occur during neurodegeneration. I will also use unfamiliar techniques, such as 2D gel electrophoresis/mass spectrometry, the switchable ER(TAM) activation system (especially in transgenic animals) and confocal microscopy. 3) The project will aid my professional growth since I will learn about mechanisms of proliferation. Knowledge of the differences between proliferating cells and non-dividing neurons, and also neoplasias in contrast to excess apoptosis, should lead to new insights into the mechanisms of proliferation (and cancer) versus cell death (and neurodegenerative disorders). I will use the excellent ER(TAM) system in particular, switchable c-Myc activity in transgenic mice means that protein expression is not lacking throughout ontogeny, leading to a powerful in vivo system, which will be of use in future research. Access to unfamiliar techniques will enhance my skills and lead to new approaches to solving scientific problems.

1) While the architecture of different neural circuits has been well documented, the behaviour of complex networks is poorly understood. How the sensory nervous system builds a representation of the surroundings through colour vision is a challenging question. I will use a behaviour assay to address this issue in the exquisitely accessible visual system of Drosophila. Flies can be trained to associate a hue of colour and a stimulus. In this context, the powerful genetic tools of flies will allow me to manipulate the opsin content of the retina as well as the ability for the photoreceptor neurons to transmit the information to higher centres in the optic lobe. I will test the effect of such alteration on the behaviour of the fly. I will then use this assay to isolate new mutations affecting colour processing. This work should help us understand how a neural network is built to integrate colour information into a tri-dimensional representation of the world. 2) For my PhD, I used a unicellular organism to study a basic cellular function, how cells switch from proliferation to differentiation. I have now shifted my focus to the actual differentiation in a complex organism. I want to study how wiring of the nervous system is established during development and how the information from the sensory cells is integrated in the brain. 3) After my extensive training with mechanistic approaches of a relatively simple process, this research plan will help me switch my interest in genetic to the different mode of thinking of developmental biology and neurobiology. The techniques used will also be different and Drosophila is a powerful system for studying problems at the organismal level.

I propose to develop a new conditional tool to study the function of specific cell types based on their local environment in an intact organism. Transgenic expression of chimeric ligand and receptor complexes on cell surfaces will modify or ablate target cells based on their contact with other specified cells. This proposal outlines a cell-contact-mediated ablation method that will exploit the Drosophila Delta-Notch signaling mechanism. The bacterial endonuclease CdtB and CRE will be expressed as inactive moieties fused to the extracellular-transmembrane portions of Notch. A second gene encoding a Notch ligand will be expressed on cells interacting with the target cells. Contact will activate CdtB or CRE by intramembrane proteolysis followed by nuclear translocation. Preliminary data from in vitro cell killing assays suggest that this system tightly and specifically controls COS-7 and N2a cell ablation. I will test the usefulness of this system using microglia and their role in prion pathogenesis as a paradigm. Activation of microglia in the brain correlates to many neuropathological disorders, including Alzheimer`s and prion diseases. However, the specific function of microglia, be it physiological, pathological or both remains unclear. Compartmentalized ablation of microglia will help elucidate the significance of this cell type in these diseases.

In the developing visual system, the spatiotemporal pattern of visual inputs play a critical role in shaping the connections of developing circuits. Using the developing retinotectal system of zebrafish larvae and combination of in vivo whole-cell recording and optical imaging techniques, I proposed to examine the development and experienced-dependent refinement of visual circuits at the cellular level. The proposal consists of four parts. In Part I, I will systematically examine the time course of synaptogenesis in the optic tectum, developmental changes in the synaptic function and in the functionally visual processing abilities, such as receptive field and direction & velocity selectivity, of the tectal neurons. In Part II, I will examine the effects of spatiotemporal visual inputs on retinotectal synapses and on the development of retinotectal map. In particular, I will examine the role of spiking-timing dependent plasticity in refinements of visual circuits. In Part III, I will furthermore examine the relationship between activity-dependent synaptic plasticity, long-term potentiation/depression, and the structural refinement at identified retinotectal connections. Finally, in Part IV, I will examine the development of zebrafish retinotectal mutants to clarify the molecular mechanism governing the development of retinotectal system. Overall, these in vivo studies will provide unique opportunities to address several fundamental issues concerning the instructive function of sensory inputs in the refinement of developing neural connections.

A major challenge of current theories of vision is to understand how the visual system performs object recognition in cluttered visual scenes. Two essential requirements for object recognition are invariance and selectivity. Neurons in primate inferotemporal cortex (IT) fulfill these requirements: they are selectively tuned to views of complex objects and their responses show significant invariance to stimulus transformations such as position changes. The aim of my project is to combine physiological, psychophysical and computational studies to investigate the dependence of IT neuronal robustness to clutter on neuron's shape- and position-sensitivities. Single-unit recordings from IT neurons in behaving monkeys will provide the first quantitative data on the relationship between shape selectivity, position invariance and clutter interference in IT. A quantitative hierarchical model of recognition will guide the experiments that will force further development of the model in an interactive and continuous process. This project will differ from my previous research experience (study of neural coding in the leech nervous system) in many aspects: the model nervous system studied - primate vs. invertebrate the neural process investigated - object recognition vs. coding of motor responses the methodology - I will learn new experimental techniques, such as neurophysiology and psychophysics in behaving monkeys the theoretical framework - I will learn to work on computer simulations of the recognition model related to my experiments. In conclusion, the proposed research will be a precious addition to my scientific development in the field of systems and computational neuroscience.