The localization and organisation of the organs in the body depends on the patterning of the Left/Right (L/R) axis during embryogenesis. Around embryonic day E7.5 in mouse, an asymmetric signal generated in the node is transferred to the left lateral plate mesoderm (this mesoderm, located in the lateral region of the early embryo, contributes to the mesenchyme of various organs). This symmetry breaking event results in the establishment of L/R asymmetric gene expression patterns responsible for asymmetric organogenesis. If an increasing number of studies focus on the regulation of these early events, less is known about how asymmetric information is translated into L/R specific morphogenetic events driving later organogenesis. The aim of this proposal is to address this question using the developing gut as a model, in both chick and mouse embryos. I propose to identify the specific molecular mechanisms at work within the cells ultimately responsible for asymmetric morphogenetic events. To this end I will first characterize, in detail, the sequence of cellular events driving gut asymmetry. I will then identify genes implicated in the control of these events using a microarray approach comparing left and right part of the gut. Finally I plan to test the implication of the best candidate genes, coming out of the microarray screening i) using genetically modified mice lines, ii) by interfering with their function using retroviral misexpression of cDNAs and RNAi constructs in chick embryos. This study will bring new insights into the molecular regulation of asymmetric morphogenesis and enlighten the regulation of morphogenetic processes commonly utilized during embryogenesis.

The aim of the research described in this proposal is to develop a physical assay for chromosome packaging and compaction in vivo using Bacillus subtilis as model system. This assay would allow us to determine the requirements for chromosome condensation in vivo and test specific models for the organization of the bacterial chromosome by proteins like Smc and Spo0J. For that purpose we will first determine the dynamic distributions of Smc and Spo0J complexes along the chromosome and their dependence on DNA replication and transcription using high-resolution ChIP on chip technology combined with cell population synchronization. This analysis will reveal the temporal and spatial regulation of Smc’s and Spo0J’s association with DNA. We will then aim at generating a conditional allele of the Smc gene that can be switched on and off within seconds using drug (FK1012 or rapamycin derivatives) dependent dimerization of FKBP12 inserted into the Smc hinge domain. Artificial hinge dimerization is supposed to block de novo loading of Smc complexes onto DNA and might thus allow us to interfere with Smc function at specific points of the cell cycle. Finally, we want to adapt and optimize the chromosome conformation capture technique (3C) for use in bacteria and detect DNA loops (juxtapositioning of certain chromosomal regions) that depend on chromosomal proteins like Spo0J and/or Smc. This assay would allow us to record the formation of chromosomal loops at different points of the cell cycle and at specific regions of the chromosome in real time. Having detected loops we could then further dissect the molecular mechanisms of Smc and Spo0J dependent chromosome organization in vivo.

Cells are composed of many thousands of different molecules that interact, create and modify each other. Many of these molecules are proteins which are in turn subject to incessant fluctuations governed by a complex interplay between their thousands of atoms. While the intricate dynamics of both layers is readily acknowledged, little is known about the interface between molecular and network-level complexity. I plan to recombine modular protein domains into artificial network motifs with defined dynamic behavior. Special emphasis will be put on the design of swapable interfaces that allow for the exchange and 'rewiring' of the different components. Modular input devices that form or break specific interactions depending on external or internal signals will be interfaced with output devices whose activity is triggered by spatial proximity between two domains. The careful control and labeling of interacting players may, in combination with cellular and molecular modeling, reveal how the dynamics and modular architecture of proteins affects the functioning of natural networks. I have previously studied the interplay of molecular dynamics and protein-protein interaction with atomic detail simulations. Through this work, I hope to complement my meanwhile purely computational background with experimental techniques, systems biology know-how and strong collaborations that allow for independent research at the cross roads of molecular and cellular complexity.

Ribosome synthesis, one of the predominant biosynthetic activities in eukaryotic cells, consumes an important part of the cellular resources and must therefore be tightly coordinated with other fundamental cellular processes. Although ribosome biogenesis has been extensively studied, the nature of this coordination and the underlying mechanisms are poorly understood. Recent results indicate that several factors required for post-transcriptional steps of ribosome synthesis are also directly involved in cell cycle progression, suggesting that these factors mediate a connection between these two fundamental processes. The aim of this proposal is to systematically identify ribosome biogenesis factors also required for cell cycle progression both in yeast and mammalian cells. Characterization of their sub-cellular localization during the cell cycle and of the protein complexes they are associated with will provide clues on their mode of action and shed light on the molecular bases underlying the potential connection between ribosome synthesis and cell cycle progression. This project is part of a new line of research I wish to develop and the HFSP CDA would provide me with an independent source of funding to start developing this project and animating my own group. The CDA would indeed allow me to finance students or postdocs and to cover the heavy cost of some of the proposed experiments.

The bandoneon mutants exhibited simultaneous contraction of bilateral muscle due to a mutation in GlyRb. Interestingly, application of strychnine, an antagonist of GlyR, to early wild-type embryos (30 h treatment at 18 hpf) not only caused bandoneon-like behavior but also chronically blocked postsynaptic aggregation of GlyRs following treatment. In contrast, strychnine treatment at larval stages (30 h treatment at 96 hpf) acutely blocked GlyRs but did not alter clustering of GlyRs. These investigations indicate that the synaptic aggregation of GlyRs is dramatically regulated by activity during embryogenesis but not at later stages. I propose to study the regulation of glycinergic synapse formation by activity and its molecular basis. The effects of activity on development of glycinergic synapses will be quantified by immunolabeling of GlyRs, physiological recording of glycinergic currents and behavior. I have found that glycinergic synapses are most sensitive to activity during the period between 18 hpf and 36 hpf. I am generating transgenic fish to label GlyRs, glycinergic pre- and postsynaptic neurons by CFP, GFP and RFP, respectively. These zebrafish will be useful to visualize the GlyRs aggregation in vivo and to isolate postsynaptic neurons by FACS. I will collect mRNA from the postsynaptic neurons and perform microarray analysis to screen candidate genes that are up- or downregulated by the treatment. I would like to focus on cytosolic protein, which can be regulated by Ca. I will examine overexpression and knockdown of the candidates to analyze their function in synapse formation. This is a new, simple model to study development and plasticity of synapse in vivo.

Mice lacking an intrinsic immune system can be transplanted with human stem cells, allowing the development of human blood cells in a manipulable system. Such a system may provide a powerful tool for developing human therapeutics. In addition humanised mice could allow a greater analysis of human lymphocyte development in vivo. Natural Killer (NK) cells comprise part of the innate immune system and are strongly implicated in the recognition and clearance of tumour cells and pathogen-infected cells. I will attempt to generate humanised mice with a fully functional and stable NK cell compartment. Firstly, I will study the reconstitution efficiency of human NK cells in newborn alymphoid mice by intrahepatic injection of human stem cells of varying phenotype (CD34+CD10+/-CD7+/-CD38+/-) and origin (foetal liver, thymus or cord blood). Transgenic-expression of human class I MHC on a murine class I MHC-deficient background and enhanced in vivo concentrations of cytokines will also be tested for improved NK lymphopoiesis. Using this system I will investigate the phylogeny of mature human NK cell subsets and characterise cell surface phenotype, tissue distribution, turnover and function. Immature NK cell development in the bone marrow and liver and commitment of human progenitor cells to the NK linage will also be studied using transfer and cell tracking techniques, as will the role of the GATA family of transcription factors in human NK cell development in humanised mice. Techniques that will be used in this project include viral infection, small interfering RNAs, adoptive transfer, cytometry, cytokine and cytotoxicity assays and numerous human graft translation procedures.

Cytokinesis, the physical separation of two cells at the end of mitosis, is a basic cellular process of immense importance: being indispensable for cell proliferation, cytokinesis is essential for the development of multi-cellular organisms. Although cytokinesis has drawn extensive research, its molecular mechanisms remain to be unraveled. The evolutionarily conserved centralspindlin complex, which consists of a mitotic kinesin-like protein and a Rho GTPase-activating protein, is a key regulator of cytokinesis. Centralspindlin localizes to the central spindle, a structure of bundled anti-parallel microtubules that forms between the separating chromosomes at anaphase. When either complex member is lost the central spindle is not formed and cytokinesis eventually fails. How centralspindlin contributes to central spindle formation and function is poorly understood. Furthermore, besides its involvement with the central spindle centralspindlin might act as a signaling platform orchestrating several molecular events during cytokinesis. To deepen our understanding of cytokinesis at the molecular level I therefore propose to identify centralspindlin-interacting proteins by drawing on both biochemical and genetic approaches. First, I will biochemically purify centralspindlin-associated proteins from mammalian cultured cells. Second, in a complementary genetic approach I will perform a genome-wide RNAi screen in C. elegans for suppressors and enhancers of the centralspindlin phenotype. Conserved interaction partners of centralspindlin will then be further characterized and analyzed for their role in central spindle formation and completion of cytokinesis.

The study of axonal growth is a central area of research in neurobiology. However, the cell-intrinsic mechanisms that regulate axonal morphogenesis remain largely to be discovered. The laboratory of Dr. Azad Bonni discovered that the ubiquitin ligase Cdh1-anaphase promoting complex (Cdh1-APC) controls axonal growth and patterning in the mammalian brain. In more recent studies, they identified the transcriptional corepressor SnoN and Id2 as key downstream targets of neuronal Cdh1-APC that promotes axonal growth. The focus of the proposal is to elucidate the mechanisms that regulate the Cdh1-APC-SnoN/Id2 cell-intrinsic pathway in postmitotic neurons and to determine how this pathway in turn controls axonal growth. To characterize the mechanisms that regulate Cdh1-APC in neurons, I will investigate the role of phosphorylation of Cdh1 and Cdh1-interacting proteins in axonal growth. To reveal the mechanisms by which SnoN and Id2 promotes axonal growth, I will determine transcriptional target genes regulated by SnoN and/or Id2, and reveal how these genes regulate the axonal growth. In addition, I would like to test the hypothesis that SnoN and/or Id2 might regulate the expression of microRNAs in neurons, because expression of several microRNAs has been recently shown to be regulated by transcriptional regulators and increasing evidence suggests that microRNAs regulate neuronal morphology. The postdoctoral research will be a great opportunity for me to learn knowledge and techniques of neuronal cell biology, signal transduction, and transcription regulation. HFSP fellowship will promote my professional growth through interdisciplinary research.

MicroRNAs (miRNAs) are short non-coding RNA molecules that post-transcriptionally regulate eukaryotic gene expression in a variety of cellular processes. Metazoan miRNAs recognise imperfectly complementary sequences in the 3' untranslated regions of target mRNAs and repress their translation by a mechanism that is poorly understood. The goal of my research project is to elucidate the mechanism of miRNA-driven translational repression. Using a combined biochemical and structural approach, I intend to dissect the molecular interactions that cause translational repression and ultimately obtain insights into their structural basis. To achieve this, I will (i) use in vitro reconstituted miRNA-induced silencing complexes (miRISCs) together with reporter mRNAs and a cell-free translation system to identify the stage of translation affected by miRNA-driven repression (ii) in parallel, biochemically test for direct interactions between miRISC and components of the translational machinery (iii) study the identified interactions by X-ray crystallography and electron microscopy. In addition, I plan to examine the link between miRNA-driven silencing and mRNA degradation in cytoplasmic processing (P) bodies, focusing on the interactions between P-body proteins and the miRISC protein Argonaute. Together, the outlined studies will bring significant molecular insights into the mechanism of RNA silencing pathways.

A remarkable feature of signaling pathways is their capacity to precisely detect and process molecular signals in spite of the highly stochastic intracellular environment. This robustness is likely the result of feedback regulations correcting for undesired perturbations. A complementary possibility that has not yet been explored is that this regulatory resilience may partially derive from the extensive proportions of signaling components that functionally overlap. For example, in Wnt pathway, the levels of cytoplasmic b-catenin are controlled by a phosphorylation complex that is stabilized by three different, yet functionally overlapping, scaffold proteins: Axin1, Axin2 and APC. In fact, while functional overlap between APC and the two AXN related proteins may be considered semantic, Axin1 and Axin2 were demonstrated functionally equivalent by knock-in experiments. In the proposed research I will test whether the regulation of Axin1, Axin2 and APC encodes for their synergistic exploitation. Specifically, I will rely on single cell measurements using polychromatic flow cytometry and real-time microscopy to test for dosage correlations between the three scaffolds. By artificially reducing levels of single scaffolds using RNAi I will test whether their functional equivalence contributes to regulatory robustness and whether scaffold redundancy in Wnt is exploited as a device for noise filtration. This study will mark the first time where redundancy is directly assessed, experimentally, for its functional advantages. Thus, apart from its importance in the context of the Wnt pathway, it will potentially demonstrates a new and previously unknown feature of genetic regulation.

Our experience of time is central to our lives. Simply shaking hands means two people have to coordinate their hands to be in the same place at the same time operating clutch and accelerator in a car requires fine timing (remember how hard that was to learn?) and even social situations require timing information - to track someone walking across a room, to interject in conversation and to decide when to leave. These simple acts we take for granted require the use of timing from milliseconds to minutes and hours. Yet the scientific investigation of temporal experience is in its infancy and lags behind our understanding of other aspects of our senses. During my HFSP fellowship I will examine the brain mechanisms of time perception using state of the art techniques of human brain stimulation (TMS) and electrophysiological recording (EEG & fMRI). By stimulating brain areas involved in time perception I will explore the temporal dynamics of early and late brain responses to temporal information and will distinguish between conscious and unconscious processes involved in timing. Studies of vision and audition have shown that perceived time and relative timing of multiple events can be distorted (e.g., flash-lag effect, chronostasis etc.). I will apply TMS to low level visual areas as well as higher cognitive areas associated with attention and memory (e.g. inferior parietal cortex) to explore the extent to which these regions of the brain contribute to those illusory time percepts. The work experiment aims to uncover specific mechanisms associated with temporal perception, and will reveal fundamental characteristics of causal interactions between different cortical areas.

Prevention of obesity is an important scientific and clinical problem, because it causes metabolic syndrome. Leptin is a key molecule that controls energy balance. Leptin over-expressing transgenic mice have lean phenotype.However recombinant leptin administration fails to cure obesity because of leptin resistance. Overcoming leptin resistance is important in therapeutic approach. 2003-Several reports suggest a link between obesity and local inflammation in adipose tissue. Although leptin is an important regulator of energy balance and has potent effect on immune cells to produce pro-inflammatory cytokines, little is known about the metabolic aspects of leptin signaling in the immune system. I hypothesize that leptin signaling in the immune system may contribute to the control of metabolic status through pro-inflammatory cytokine secretion. Thus leptin sensitivity of macrophages may affect the inflammation of the organism, which would affect the metabolic status. I propose to address this possibility by analyzing the effect of leptin sensitivity of immune cells in mice. In this context, I will generate bone marrow chimera mice lacking leptin signaling in immune cells. And I perform in vivo analysis of leptin sensitivity. To gain insight into the effect of leptin on T cells, I will perform a microarray analysis of T cells treated with leptin in vitro, in order to characterize the effect of leptin on gene expression and to identify key gene products involved in coupling between the immune system and the metabolic status. If this hypothesis turns out to be correct, it would provide an important advance to our understanding of the links between inflammation and metabolism.

During assembly, viruses must package their newly replicated genomes for delivery to other host cells. In many viruses carrying double-stranded DNA, the genome is translocated into a preformed capsid [2]. The process of DNA translocation is driven by ATP dependent molecular motors that generate the force required to pack the genetic material into protein capsids that are barely large enough to enclose the genome. We propose here a detailed study of the mechanism of packaging in the bacteriophage phi-29 portal motor, by exploiting single-molecule manipulation and single molecule fluorescence techniques. We will study the mechanism of coordination of the different ATPase subunits in the motor and the coordination between the chemical and mechanical cycles. Next, we will focus on the conformational changes involved in packaging (both on the packaging motor and on the packaged DNA). We will then attempt to determine the role of pRNA in the motor mechanism. Finally, we will study the interaction between the motor and the substrate. This single molecule approach offers the possibility of dissecting the complex coordination involved in the packaging process and possibly shed light into the molecular mechanism of a number of ATPase-based ring translocases.

Understanding the neural circuits that drive behavior and the genes that function in these circuits is a fundamental aim of neuroscience. Drosophila is particularly amenable to such study and many innate behaviors, courtship in particular, have been well defined. When a male fly encounters a receptive female, it performs a stereotyped mating ritual that includes orientation, tapping, singing, licking, and finally copulation. The molecular pathways encoding for sex-specific behavior have been heavily studied. Most evidently, the fruitless (fru) locus encodes for fruM, a male specific transcription factor that is expressed in approximately 2% of CNS neurons and acts as a genetic “master gene” controlling male courtship behavior. Though fruM has been assigned a role in courtship, little is known of the genes and neural circuits that mediate communication between the two sexes and drive specific components of this ritual. I will focus on one of these components: the courtship song the male produces to seduce the female. This is a particularly attractive system to study because, unlike the chemosensory signals that drive other components, the relevant acoustic signals are well defined and readily manipulated. In addition, the analysis of the courtship song can be partly automated. By combining two genetic technologies used for tissue specific expression in Drosophila, GAL4/UAS and FLP recombinase/FRT, I will selectively manipulate subsets of fruM expressing neurons and test flies for song defects. These experiments will allow for the dissection of fruM expressing neurons that control male courtship behavior to identify circuits specifically involved in song.

Development of functional neurons from embryonic neuroepithelium is mediated by series of events including specification into multipotent neural precursor cells, commitment to differentiated neurons, and maturation into functional neurons composing of neural network. These processes are tightly controlled by intrinsic genetic programs, but also affected by environmental condition. However, the extent of the external signals that can modify the intrinsic programs is not clearly addressed yet. My previous studies identified an intrinsic program involves in glial fate specification against default neural fate of optic neuroepithelium. Vax homeotic transcription factors were critical player for glial fate acquisition of optic neuroepithelium under reciprocal antagonism between Shh and protein kinase A (PKA) signaling pathways. My current researches are aiming to the understanding roles of intracellular signaling pathways such as PKA and phosphoinositide-3 kinase (PI3K) pathways, which are cross-talking to many external developmental signaling pathways in neurogenesis of vertebrate retina. The strength of the signaling pathways will be genetically modified in mouse retinal progenitor cells (RPCs) to investigate the roles of intracellular signaling pathways as central modulators of intrinsic genetic programs upon the changes of external cues.