It is becoming increasingly clear that the information encoded in the linear DNA sequence is insufficient to fully account for the complex regulation of genes and genomes. Higher-order chromatin structure and epigenetic regulation via chromatin modifications are now recognized to be important mechanisms of gene regulation. An additional emerging mechanism of genome regulation is the control by higher-order organization of chromatin and chromosomes. The aim of this project is to reveal how higher-order structure of chromatin and chromosomes changes during neural cell differentiation and how nuclear architecture affects expression of gene programs. I will focus on uncovering how extra-cellular cues affect gene expression programs and differentiation in the nervous system. Specifically, I will investigate the spatial positioning of chromosomes and cell-type specifically expressed genes, and analyze kinetics of proteins and gene loci in neural stem cells and during their differentiation into neurons and astrocytes. This project takes advantage of my expertise in neural differentiation and will enable me to acquire training in new methods and a novel field of research. At the end of the fellowship, I will have technical skills in advanced imaging and expert knowledge in the area of spatial genome organization, and this will allow me to explore further the complex mechanisms of cellular differentiation in the nervous system. I believe that postdoctoral training in the field of nuclear architecture in Dr. Misteli’s laboratory will ideally complement my expertise in molecular biology and will prepare me for a career as an independent scientist.

Small non-coding RNAs play an unexpectedly large role in regulating gene expression. small interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate both specific gene expression and more globally regulate chromatin structure and genomic stability. Both siRNAs and miRNAs act via the RNA interference (RNAi) pathway. At the heart of RNAi is a protein-small RNA complex, the RNA-induced silencing complex (RISC) that catalyzes target RNA destruction. Several proteins in the RISC have been identified, and many of these are required for metazoan development. My research seeks to dissect the pathway of RISC assembly and the mechanism of RISC action. I am especially keen on understanding what differentiates the RISC function in target cleavage (RNAi) from its role in translational repression via miRNAs. My previous experience dissecting the mechanism of tRNA-maturation will serve me well in this pursuit, but my study of the RNAi pathway will also give me new experience with Drosophila developmental biology and genetics. Thus, a HFSP fellowship would help me to grow beyond my biochemical expertise and will promote my professional growth through interdisciplinary research.

Synapses are precisely assembled and organized during brain development. Despite the central role of synapses in neuronal functions, mechanisms underlying synapse formation remain unclear. The primary purpose of this project is to define molecular mechanisms for synaptic development and the relationship between synaptic morphology and synaptic function. I will focus on the role of stargazin, which is the mutated protein in an ataxic and epileptic stargazer mutant mouse and is involved in synaptic targeting of AMPA receptors. I will identify novel stargazin-interacting proteins by taking advantage of previous my biochemical techniques. Next, I will investigate the functional roles of the stargazin-interacting proteins by taking advantage of the host laboratory’s experience in neurobiological and neurophysiological approaches, which are indispensable for achievement of the proposed project. This research with Dr. David S. Bredt at UCSF, who is one of the world leaders on the mechanisms of postsynaptic protein assembly, will provide a large step toward my scientific goal to address fundamental questions in brain science. This project will also provide me with invaluable training in the discipline I plan to pursue as an independent scientist upon completion of the fellowship.

Kinesins are essential for microtubule-dependent cargo transport. While the roles of all six budding yeast kinesins have been explored, we lack a comprehensive understanding of which kinesins transport specific cargo in a single higher eukaryote that typically contains more than 20 kinesins. Furthermore, there has been no comprehensive view of the mitotic roles of kinesins in a single cell line of higher eukaryotes. I plan to analyze these issues in Drosophila S2 cell line. At first, I will perform RNAi against all kinesins in S2 and observe defects in mitosis by microscopy.

1) Goal of the project is to address the role of aminophospholipid translocating ATPase (ATPase II) in neuronal functions, especially in the exo-endocytosis of synaptic vesicles (SVs). ATPase II is a member of a subfamily of P-type ATPases and is thought to play a role in the maintenance of membrane phosphatidylserine (PS) asymmetry in certain cellular organelles. There is indirect evidence that PS translocation is important for the exocytosis of secretory vesicles and genetic studies have demonstrated a functional interaction of the yeast homologue of this ATPase (Drs2) with clathrin and with the endocytic protein Pan1. ATPase II is especially rich in brain and my preliminary experiments indicate that ATPase II is specifically concentrated on SVs. The project will address the role(s) of ATPase II in neuronal function and will focus on its role in exo-endocytotic cycle of SVs. 2) My previous research experience has focused on the characterization of synaptic-like microvesicles in endocrine cells. As part of these studies I have identified vesicular glutamate transporters in vesicles of peripheral tissues. I have mainly used immunohistochemical and electronmicroscopical techniques. In this proposal, I want to clarify novel aspects of exo-endocytotic cycle of SVs, and I plan to familiarize myself with biochemical and molecular approaches. 3) I believe that broadening my technical and intellectual expertise in a top laboratory will be essential to complete my training toward an independent career in Neurobiology. I also believe that the field at the interface between lipids and membrane traffic is a promising area which is likely to blossom over the next few years.

I have studied mouse neural development by focusing on transcription factors that regulate neurogenesis. Molecular biology and reverse genetics techniques such as gene targeting and viral misexpression were useful to clarify the function of transcription factors in embryogenesis. During the course of the above research, I have got interested in the mechanism by which the complex neuronal networks are generated. I propose a genetic analysis of the development and function of neural circuits regulating a simple behavior in zebrafish. The earliest locomotion behavior exhibited by developing zebrafish is spontaneous coiling of the trunk. There appears to be two different behaviors. The earlier coiling is independent of the brain and appears to require electrical activity of spinal neurons but not synaptic transmission. The later coiling requires input from the hindbrain and synaptic transmission within the spinal cord. I will examine the earlier coiling by ramp assay, which distinguish the mutants defective in slow coiling. The later coiling defect exhibits curled trunk by dechorionation at 30 hpf. Screening by these assays should identify coiling mutants. I will directly examine these behaviors by recording with a CCD camera. I will also examine both the morphology (immunostaining and dye labeling) and physiology (Calcium imaging and patch clamp recording) of spinal and hindbrain neurons involved in coiling behaviors. Possible simple circuits of zebrafish neuronal network would be suitable for detailed analysis of vertebrate behavior. With the above research project, I hope to extend my research career to zebrafish genetics and neurophysiology to be a more creative researcher.

The proposed post-doctoral research is to characterize Arabidopsis SPA1 which is involved in PhyA signaling, especially, to identify the biochemical mechanisms of action of SPA1 in the far-red light signaling. Based on some previous data, it is postulated that one function of SPA1 is to regulate the activity of COP1, which acts to repress photomorhogenesis in the absence of light and directly interacts with SPA1, as a ubiqutin E3 ligase. To elucidate this hypothesis, I propose to carry out two series of experiments on SPA1. In the first, I will examine the effects of increasing the concentration of SPA1 on both auto-ubiqutination in vitro and determine the subcellular localization of SPA1 to investigate possible protein co-localization with COP1 in plant cells. In the second, to examine the function of SPA1 gene in Arabidopsis, SPA1 gene will be placed under the control beta-estradiol inducible promoter and introduced into wild type, phyA and cop1 mutants of Arabidopsis and the phenotype will be examined under various light conditions in the absence or the presence of the inducer. In addition, I will screen T-DNA-tagged Arabidopsis plants for SPA1 homologs mutations and examine the phenotype by the insertion of T-DNA. These experiments should dissect the role of SPA1 in PhyA signaling. As described above, the proposed post-doctoral training will allow me to use a combined genetic, molecular and biochemical approach to elucidate an important problem in plant development in response to light, the role of PhyA-signaling, although my doctoral training has mainly been in plant cell biology.

In different environments, genetically divergent populations tune their way of life to compete successfully for resources. While variation across large phylogenetic distances is representative of macroevolution, polyphenisms within a species or population are likely to be the most abundant elements to drive evolution. Studies addressing the genetic basis of phenotypic variation within populations will therefore provide novel insights into the mechanisms of biodiversity. The central focus of this proposal is to understand the molecular relationship between sexual selection (female mate preferences) and the male body-color patterning in guppies, Poecilia reticulata. This tropical fish has been one of the most important species to study the relationship between “intra”-specific (within a species) natural variation and adaptive traits, including sexual selection, an important question in evolutionary theory. However, the molecular basis of this variation is completely unknown. This project aims to lay the foundation for understanding sexual selection in guppies as a model study for quantitative evolutionary genetics.

In C3 plants, CO2 is fixed by RubisCO. At the atmospheric CO2 concentration, oxygenase reaction of RubisCO, an energy-wasting process, is inevitable. In C4 plants, CO2 is fixed to form a C4 compound, which is transported to the bundle sheath cells (BSC), where it is decarboxylated by malic enzyme. Resulting release of CO2 maintains the CO2 concentration high enough to eliminate the oxygenase reaction. Since the malic enzyme reaction also provides NADPH, ATP is preferentially required from the light reaction in BSC. A physiological question how the ATP/NADPH production ratio is controlled has not been answered at a molecular level. I have studied molecular genetics and recently identified a gene responsible for cyclic electron flow around photosystem I in C3 plants. Highly probably, C4 photosynthesis utilizes such cyclic electron pathways around PS I in BSC, but molecular component have not been described so far. Recently, C4-like photosynthesis was discovered in stem and petiole cells of C3 plants and it seems possible that C3 plants can also manage tissue-specifically C4 photosynthesis. I focus on the control of ATP/NADPH production during C4 photosynthesis, by extending my research background from genetics to physiology and biophysics. For this purpose biophysical techniques (photoacoustic, fluorescence imaging), only available in a few laboratories including the host laboratory, will be used to monitor in vivo electron flow activity in a C4 plant (maize) as well as in specialized cells of a C3 plant (tobacco) able of C4 photosynthesis. I feel extremely important to extend my research area by establishing a bridge between current molecular genetics and physiology.

Regulated protein degradation plays an essential role in the development of all organisms. In particular, ubiquitin-mediated proteolysis has recently emerged as a central regulatory mechanism for many cellular processes. In this system, a poly-ubiquitin chain serves as a degradation tag, causing destruction of tagged substrate proteins via proteolysis in the 26S proteasome. This process is catalysed by ubiquitin-activating (E1), -conjugating (E2), and ligase (E3) enzymes. Generally, E3 ubiquitin ligases determine the specificity of substrate ubiquitylation. Recently, a new family of E3 ubiquitin ligases containing a RING finger-like domain called U-box has been identified. The Arabidopsis genome contains a large number (~63) of genes encoding U-box proteins (referred to as PUBs for Plant U-Box). However, to date, only a few U-box proteins have been functionally characterized. In this proposal, we test the hypothesis that specific Arabidopsis PUB proteins act as E3 ligases in signalling associated with the the phytohormone gibberellin (GA). The GA signaling pathway is emerging as an important paradigm for the functioning of the ubiquitin-proteasome pathway in plants. GAs are essential regulators of plant growth and development, and GA-deficient mutants exhibit a dwarfed, retarded growth phenotype. GA promotes plant growth by overcoming the growth-repressive effects of a family of nuclear repressor proteins known as the DELLA proteins, and GA-induced proteolysis of DELLA proteins is a key step in GA signaling. This proposal focuses on the relationship between Arabidopsis PUB proteins and the DELLA proteins. Considering the relatively high sequence conservation of the U-box domain among eukaryotes, this research will provide a good prototype for the understanding of U-box protein function in eukaryotes in general, and not just in plants. In addition, this work will provide important new information that is essential for a complete understanding of the molecular mechanisms of ubiquitin-mediated proteolysis in plants, particularly with respect to its role in plant hormone signaling pathways.

Calcium is an important signaling molecule that modulates various cellular functions including gene expression and synaptic activity. We have identified and characterized a calcium/calmodulin-dependent protein kinase (CaM-K) cascade which is activated by calcium/calmodulin (CaM). CaM-kinase kinase (CaM-KK) phosphorylates and activates CaM-kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV) as well as protein kinase B (PKB). In the present application, we will use a method for in vivo identification of CaM-KK signaling complexes using TAT-mediated protein transduction into the brain and the Tandem Affinity Purification (TAP) coupled with mass spectrometry. The TAT-mediated protein transduction method is a technique to introduce purified proteins into cells as well as into the brain of rodents. The TAP method is a protocol for purification of endogenous protein complexes under native conditions. Combining these two techniques, we propose to purify proteins complexed with CaM-KK from brain under physiological conditions. We will identify members of these CaM-KK protein complexes by mass spectroscopy and investigate the physiological regulation of these interactions as well as their effects on CaM-KK activity and subcellular localization. I have been extensively trained in the area of developmental biology. I want to research experience in different areas to expand my scientific background. The studies proposed in this application are not simply a change in research area, it will give me the diverse background and experience of modifying a new methodology. These experiences will greatly broaden my technical abilities and allow me to pursue my scientific interests.

1) At early stages of development, the heart is a single tube. In lower vertebrates such as fish, it remains as a two-chambered linear tube, but in higher vertebrates such as mammals, the heart is divided into four chambers (left/right, ventricles/atria) separated by septa. The morphology of each chamber is specific to its individual function, and the separation of chambers into left and right components is required for separate pulmonary and systemic circulations. A few molecules are expressed in a polar fashion in the heart, but the molecular mechanisms that specify the identities of each chamber and the positions of septa have remained unclear. My project is to find the molecular mechanisms of chamber formation during cardiovascular development with as aim to explain the evolution of the vertebrate heart from a simple two-chambered structure to a four-chambered organ adapted to dual circulation, and how this organ forms aberrantly in human disease. 2) So far I have studied limb, eye and heart development using chick embryos. Chick is very useful to study morphogenesis, because methods of in ovo manipulation have been established. However in chick genetic methods have not been established, and it develops differently from mammals. I am interested in human disease, and I hope to apply my findings to therapy of heart disease. 3) My project allows me to learn advanced mice genetics and microarray techniques. I will be able to develop new methodologies to study mouse embryology by combining my manipulation techniques with mouse genetics. The study of the heart is still unexplored, and this will allow me to expand my research horizons towards therapy of human disease.

Electrophysiological analyses and optical imaging have been used to investigate excitable cell firing and calcium transient in acute brain slice, cultured brain slice or cultured neurons, but both techniques are incapable of recording from multiple neurons with single-cell resolution in vivo. To overcome this limitation, we propose to introduce genetically-encoded neural activity probes such as Yellow Cameleons, Clomeleon or Flash to a population of cortical neurons by in vivo electroporation technique and measure ensemble of neural activity with two-photon microscopy. Two-photon microscopy has enabled fluorescence imaging in the intact brain of freely-moving animals. This approach will establish the method to image neural activity of alive animals during a behavioral tasks. I will utilize the rodent somatosensory barrel cortex as a model system, which provides us with a good opportunity to analyze how the sensory stimuli are processed by neural networks. There are many accumulated information from previous works of electrophysiological analyses and optical imaging, and I can compare my data with preivious results. My graduate and postdoctoral training were concerning mammalian developmental biology. My background of developmental biology, mouse genetics and molecular biology allowed me to pursue this goal. Especially, in vivo electroporation technique is indispensable for this project, which is an efficient method of gene delivery specifically to bulk of specific subtypes of mature neurons and not to glial cells. A HUMAN FRONTIER SCIENCE PROGRAM would allow me to develop and expand my research interests in the information processing of mammalian cortex.

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.