I will study the importance of Transforming Growth Factor beta (TGFb) signalling for the regulation of embryonic cell proliferation. A mutation in a receptor for drosophila Activins, the least probably least studied TGFb ligands, specifically impairs cell proliferation. I will therefore genetically analyse the importance of different Activin signalling components for the control of cell division. As excessively proliferating tumour cells present alterations in cellular organisation, I will study trafficking and organisation of the Activin signalling machinery and determine its importance for the regulation of cell proliferation. I will analyse the organisation of zebrafish Bone Morphogentic Protein signalling to determine the importance of cellular architecture for vertebrate TGFb signalling in epithelial and mesenchymal tissues. Finally, I will use drosophila and zebrafish to study the function of SARA, a TGFb signalling adapter that associates with the central spindle during mitosis, suggesting a potential link between TGFb signalling and the mitotic apparatus. By studying proliferation control, I want to tackle a major aspect of development which I have not considered previously. Training in cell biology and drosophila genetics will considerably widen my methodological playfield. The experience of this fellowship will therefore be both different from and complementary to my previous research. Beyond the period of the fellowship, the ability to take advantage of both drosophila and zebrafish, two major model organisms, and the acquired competence in cell biology will allow me to work in the exciting new field at the interface between cell and developmental biology.

Vesicle trafficking of receptors has been recognised as an integral part of signalling in animals. In plants, BRI1-receptor mediated signalling is one of the best understood to date. However, nothing is known about the interplay between signal perception and regulation of BRI1 transport. I propose to investigate BRI1 trafficking and its importance for signal transduction. BRI1-GFP is detected in root cells, well-suited for cell biological manipulations. Many non-lethal brassinosteroid (BR) mutants are available and facile Arabidopsis transformation protocols will allow me to screen for PCR-mutagenised BRI1 trafficking mutants in defined genetic backgrounds. This will be done in-planta, with proteins that are stably expressed to endogenous levels. The combination of genetic, molecular biological and cell biological tools will give new insights into brassinosteroid signalling and possibly establish Arabidopsis as an attractive model for investigation of receptor cell biology. I acquired good skills in molecular genetics and cell biology. However, I never undertook mutant screens and only occasionally utilised live-imaging with GFP-fusion proteins. Nonetheless, I have precise ideas of the advantages and limitations of using fluorescent proteins. At the Salk I will find all the necessary equipment and I will be in a group with large experience in genetic screens and BR biology. I will be able to build on my skills, but also to explore new imaging and cloning techniques and to establish new mutagenesis methods in Arabidopsis. In the future I hope to exploit this new mutagenesis approach and to further dissect the interplay of endosomal trafficking and signal transduction.

MicroRNAs are evolutionarily conserved non-coding RNAs of ~22 nucleotides (nt) that have emerged in recent years as regulators of gene expression and development. I propose to study the role of miRNAs during vertebrate morphogenesis using zebrafish as a model system. I will first take a combined biocomputing and cloning approach to identify miRNAs and their targets. Using gain- and loss-of-function approaches, I will then perform a screen to identify miRNAs that orchestrate early development and organogenesis. Finally, I will focus on a small subset of miRNAs to determine how their targets control development. During my previous research I addressed how the Wingless morphogen gradient is established and how it regulates tissue growth, using Drosophila as a model system. This project provided new insights into the regulation of signaling pathways during development and gave me a thorough training in Drosophila developmental genetics and signaling. My proposed postdoctoral project shifts my focus to the study of RNA regulation during zebrafish development. Hence, I will change both my area of research and the model system of choice. My proposed project will be a new, independent line of research in Dr. Schier's laboratory and employ a novel combination of molecular biology, in vivo imaging, phenotypic characterization and biocomputing approaches. The expertise in vertebrate developmental genetics and zebrafish biology in Dr. Schier’s laboratory will provide an excellent training and research environment. A HFSP fellowship would allow me to broaden my scientific training and establish an independent line of research that will form the basis of my future laboratory.

1) The nuclear envelope (NE) serves to compartmentalize the nucleus and cytoplasm of higher eukaryotic cells. In multicellular organisms the stable NE is dynamically rearranged during cell division. Since the precise molecular mechanisms of NE reformation at the end of mitosis are still unknown, identification of novel proteins involved in this process may provide us with valuable information on how it takes place. Caenorhabditis elegans is a powerful multicellular experimental system to study such phenomena. By entire Caenorhabditis genomic RNA-mediated interference (RNAi) screen several novel genes with possible function in NE assembly have been identified. I plan to determine, which of these new candidate genes have real function in NE assembly, and then characterize the selected genes using C. elegans embryos as in vivo and Xenopus egg extract as in vitro system. Combination of the results got with the above-mentioned experimental systems might be of great potential in describing the molecular mechanism of NE assembly. 2) In the proposed research work I intend to use new in vivo (C. elegans) and in vitro (Xenopus egg extract) experimental systems, which differ from the system (Drosophila) that I have used until now. Furthermore, I also plan to use many modern biochemical techniques and approaches, which are new for me. 3) Experiences reached with the proposed research work as well as with the mental training in the outstanding laboratory of Prof. Mattaj, together with my previous research experiences, will broaden my intellectual horizon and help me to be prepared for new challenges and to solve the critical scientific questions in the future.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one pathway of the ubiquitin (Ub) system, which was extensively studied by the Varshavsky laboratory over more than two decades. I propose to carry out experiments whose chief subject are the Ub ligases (E3 enzymes) of the UBR family that mediate the N-end rule pathway in both the yeast Saccharomyces cerevisiae and the mouse. The aims are to identify new physiological substrates of this pathway and to dissect its regulation in yeast. The understanding thus obtained will be relevant not only to this pathway but also to the Ub system at large. My graduate research comprised an extensive study of metabolic pathways using biochemical approaches. In my postdoctoral work, I intend to complement this experience by learning, in the Varshavsky lab, the techniques and approaches of molecular genetics as applied to the N-end rule pathway, a universally present proteolytic system. Working with yeast and mammalian cell cultures is also new to me. The proposed studies will, therefore, allow me to broaden my knowledge of eukaryotic biochemistry and genetics. In sum, the proposed research fundamentally differs from my graduate research experience. Carrying out the biochemical and genetic dissection of the N-end rule pathway, and studying the targeting of protein substrates to the proteasome will expose me to a broad range of methods and concepts that I did not encounter, thus far, in my graduate research, which was focused on protein physical chemistry and enzymology. The proposed projects are therefore crucial to my further development as a scientist.

Recent studies reveal that mammalian stem cells (SCs) are more versatile in choosing fates than previously appreciated. This property holds tremendous promise, particularly for less controversial adult SCs, in tissue regeneration. Challenges that remain unsolved include the relation between adults and their embryonic counterparts, and the mechanisms regulating renewal and lineage determination. Skin epithelium provides an excellent model for investigating these problems, since it can be maintained and propagated in culture. My research focuses on isolating and characterizing SCs, enabling me to explore how self-renewal is achieved and to identify mechanisms involved in regulating lineages. After isolating multipotent epithelial SCs and their committed progeny from mouse skin, I will compare their programs of mRNA expression, placing emphasis on known markers of hematopoietic and other SCs, including cell cycle and chromatin structure genes that might provide clues to the slow-cycling properties of SCs. Real time PCR, subsequent in situ and immunofluorescence will reveal which markers are expressed in the skin niche, and how these patterns change with commitment. The most interesting of these genes will be explored by RNAi in vitro and in vivo first and eventually by transgenic technology. My prior training has focused on cell-cell communication in a tissue in Drosophila. Through my post-doc, I hope to understand the complex mechanisms controlling SC self renewal and fate decisions in skin, and similarity between adult and embryonic SCs. This training will be important for my career goals as a professor of Cell and Developmental biology focusing on SC research.

SERA5 is emerging as a key protein in the growth of the blood-stage cycle of the human malaria parasite P. falciparum, but its precise biological function remains unknown. Current evidence implicates a role for SERA5 most likely in the rupture or invason of infected red blood cells. In this project I aim to determine the function of SERA5 and in doing so establish a powerful new genetic technology of broad applicability to the malaria field. I propose to delete the gene encoding SERA5 using a novel conditional knockout approach, which will provide insight into its function and its relationship to downstream cellular pathways and processes. Furthermore, I plan to determine the localization of SERA5 and its downstream substrates, which will help unravel its role in the rupture/invasion pathway. Ultimately, understanding invasion will aid in the discovery of new ways to therapeutically intervene and prevent malaria. To achieve the goals of this proposal, I will study the key molecular pathways and interactions that a protease must make in the process of malarial rupture/invasion. My prior work involved the development of chemical probes and inhibitors to understand the overall function of a protease, but not the molecular details. To accomplish these new goals I will need to learn the combined use of a host of genetic and molecular biology tools with cell biology and proteomics analysis. This fellowship will allow me to add powerful genetics techniques to my skill set as well as a deeper understanding of malaria biology.

In mammals, dosage compensation of X-linked genes between sexes is achieved by X-chromosome inactivation in female cells. The Xist gene, which encodes a non-coding RNA is located on the X-chromosome and is required and sufficient for X-inactivation. At the onset of X-inactivation Xist expression is upregulated on the future inactive X-chromosome (Xi), and the RNA associates with chromatin to coat the entire chromosome in cis. The spread of Xist RNA along the chromosome correlates closely with silencing of the inactive X-chromosome. Although the mechanisms by which Xist RNA orchestrates X-inactivation are largely unknown, it has been demonstrated that the chromatin of the Xi accumulates epigenetic modifications upon transition through the stages of X-inactivation. Xist RNA could act as a scaffold that directs chromatin-modifying activities in a defined spatio-temporal manner to mediate the ordered series of epigenetic modifications required for the progressive hetero-chromatinization of the Xi. These large scale and uniform changes of histone modifications on the Xi provide a powerful system to study facultative heterochromatin formation. Here, I propose to study different stages of the X-inactivation process, including the mechanisms of choice, enactment and maintenance of X-inactivation. These studies will help to understand how Xist RNA spreads along the Xi, and how Xist RNA mediates silencing, elucidating the mechanism of X-inactivation and obtaining a more complete picture of the interplay between RNA and chromatin modifiers and hence provide insight into the epigenetic regulation of gene expression in mammalian cells.

Developmental mechanisms of differentiation can be divided into two types: the generation of distinct cell types from a single multipotential progenitor cell, and the sequential stages in the process of generating a specific cell type. B cell development, as do development of other cells of the hematopoietic system, offers a unique opportunity to study both mechanisms. B cell development can be divided into several stages: commitment to B cell development, progenitor B cells (pro-B), pre-B cells and mature B cells. The transcription factors involved in early B cell development are relatively well characterized, but less is known about later stages of this development. I propose to study the mechanisms involved in pre-B cell stage to B cell transition. I will use a knockout mouse described recently at Dr. Singh's lab. These mice have had IRF4,8 deleted, causing a pronounced pre-B cell stage arrest, which offers a unique opportunity to study this cell stage. I will first characterize the molecular phenotype of IRF4,8-/- pre-B cells, especially what is preventing light chain rearrangement, a major event that should occur at this stage. Then I can use these mice to identify genes involved in pre-B cell stage to B cell transition, and to analyze the temporal order of events in this transition. My work until now had been to study how one factor - DNA methylation - affects gene transcription. In this work I will study how different transcription factors work together to create the correct gene transcription pattern. This combined knowledge - of how single elements work, and how different elements work together, will enable me to understand complex biological systems.

I propose to study the serotonergic modulation of C. elegans locomotory behavior using optical activity recording and optical stimulation of specific neurons together with pharmacological, molecular and genetic tools. A genetically-encoded voltage sensitive probe will be expressed under specific promoters in sensory and interneurons and used to record neuronal activity. This will enable the identification and characterization a postulated locomotory CPG and permit characterization of network activity under various conditions. A transgenic photostimulation system will be also expressed in specific sensory and interneurons to stimulate them in a noninvasive manner. The effect on the nematode behavior and neuronal activity will elucidate the locomotory network and its functional connectivity. Pharmacological and genetic manipulations will be used to dissect different aspects of the 5-HT modulation of behavior. Lastly, a naturally occurring 5-HT hypersensitivity allele will be cloned. Apart from my 3-month long Grass Fellowship, I had never studied C. elegans, applied molecular techniques nor used activity imaging. This is a good opportunity to fill the molecular and genetic gap in my scientific background. Also, I will be able to take these optical tools for recording and stimulating neuronal activity in C. elegans and use them when I start my own laboratory and share them with colleagues. C. elegans is a very promising neuroethological model. In several years it will probably be the first animal in which all neurons will be recorded at once during natural behavior and behavioral plasticity.

Humans, more than any other primate, are first and foremost social creatures. Yet we still have little understanding of the neural basis of social cognition and behavior. The research proposed here belongs to an emerging area of interdisciplinary research, social neuroscience. My aim is to develop new techniques for studying social behavior, based on brain imaging (fMRI). Specifically, I wish to search for neural correlates of processes such as social imitation, social skills and the implicit and explicit effects of contextual factors on our social behavior. In my PhD I studied the organization and function of high-order visual regions, under highly controlled and exceedingly simplified settings, while treating each subject in isolation. However, our most basic human traits - our thoughts, goals, intentions and feelings - are influenced by our social and cultural environment. In my last PhD project I therefore developed methods to study brain activity with more natural stimuli and viewing conditions, and showed their validity by replicating results from my previous work. I propose to use this "natural viewing" paradigm to study the pathways by which social processes influence and are influenced by brain function. The development of a new field, especially one that spans disciplinary boundaries, takes time. My work will advance collaboration between three leading scientists in social psychology and neuroscience. I believe that the proposed project, though ambitious, is feasible with this mentoring team, and will result in a better understanding of how the brain performs its social functions.

A longstanding aim in neuroscience is to find neuronal substrates of behaviors. Here, we (i) explore a novel strategy to identify and functionally characterize the neuronal circuits involved in specific behaviors in genetically modified mice and (ii) apply this method to investigate the role of the Amygdala central nucleus (CeA) in innate vs. conditioned fear. We propose to use genes specific for neuronal populations that show behavior induced activity, to express genetically encoded trans-synaptic tracers to map the neuronal circuitry the neurons are part of. Similarly, expressing an inducible genetically encoded neuronal silencer under control of the same genes will allow silencing of this circuit in behavioral paradigms for innate and learned fear. Thus, we hope to functionally dissect the role of the CeA in these two forms of fear to better understand the neuronal basis of anxiety disorders in human. The proposed research will extend my training to behavioral neuroscience and thus complements my previous experience in functional and developmental neuroscience. Both topic and approach, will be central to my future research outlined previously. Future directions, using similar strategies that combine molecular genetics, genomics and behavioral neuroscience, are (i) the identification and functional characterization of genes – associated with altered brain function in evolution or disease - and (ii) to exploit across species gene expression data (human, chimp, mouse) to identify homologous neuronal structures in mice (even if they cannot be defined anatomically) and then address human specific traits in the mouse as model system.

Meiosis I is fundamentally different from meiosis II and mitosis in that homologous chromosomes, not sister chromatids, are segregated to opposite poles. By using fission yeast as a model system, I want to study the mechanisms of this unique chromosome segregation. I will carry out two genetic screens to find novel factors that are involved in setting up the specialized chromosome segregation of meiosis I. Specifically, I want to know what allows sister kinetochores to be oriented towards the same, and not opposite spindle poles during meiosis I, and I want to identify genes required for sensing and translating chromosome attachment errors typical for meiosis I. The evolutionary conserved Aurora kinase is a candidate for being involved in the latter process, and I will therefore also examine the function of this protein kinase in meiosis.