MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally control gene expression by targeting mRNAs for translational inhibition or degradation. They exert essential functions during embryogenesis, developmental timing, differentiation, cell growth and apoptosis. MicroRNA genes are under control of RNA polymerase II promoters enabling tightly regulated stage- and tissue-specific miRNA expression. Deregulated expression of miRNAs has been implicated in human cancer. This study aims to unravel the transcriptional network regulating miRNA gene expression in Caenorhabditis elegans. The specific aims within this research project are: (1) Identification of transcription factors regulating miRNA gene expression. Transgenic animals will be generated carrying miRNA promoter-GFP transgenes that mimic endogenous miRNA expression. I will use these strains to screen an RNAi library comprising known and predicted C. elegans transcription factors in vivo. Candidate transcriptional regulators of miRNA expression will be verified on endogenous miRNA genes. (2) Identification of transcription factor binding sites in the C. elegans genome. For this, I will perform Chromatin Immunoprecipitations of FLAG-tagged transcription factors derived from (1) and identify their promoter binding sites using commercial C. elegans genome-wide tiling arrays and a direct qPCR approach. (3) Analyses of genetic interactions between transcription factors and miRNAs. Epistasis experiments will be performed using C. elegans miRNA mutant strains along with an RNAi approach. This study will constitute the first systematic genetic screen to identify miRNA transcriptional regulation in any organism.

Protein trafficking pathways in eukaryotes, their regulatory mechanisms and the molecular machinery involved are relatively well understood. However, the underlying genetic regulation has been largely overlooked. The question how cells adapt to various trafficking requirements, and to which extent this regulation is controlled transcriptionally is of great importance to the field of cell biology. I will address this question using Arabidopsis seed storage protein trafficking. During seed development, large quantities of storage proteins are transported into a novel organelle, the storage vacuole. This process appears to be genetically tightly regulated, as it occurs only during this stage of the plant's life. Genes involved in these events have been identified in the host laboratory and other groups. I aim to identify their transcriptional regulation to decipher common cis-regulatory elements, their transcription factors and/or epigenetic regulators. The identified regulators will be first analyzed for functional significance in storage protein trafficking. Further analysis will 1) provide better understanding of the role of single factors and the extent of transcriptional networks required for the complex cellular rearrangements involved in storage protein trafficking, 2) answer the fundamental question whether all these responses are developmentally programmed, or whether cells can actually sense their current transport requirements, 3) contribute to our understanding of such mechanisms in other eukaryotes and 4) help identifying the main regulators of storage trafficking to alter the storage capacity in non-seed tissues, an important trait in plant biotechnology.

With each mitotic division cells confront a critical challenge of accurately duplicating and dividing all essential cellular components. Understanding the various molecular machineries that enable this partitioning is therefore of fundamental importance in cellular biology. The Endoplasmic Reticulum (ER) is a large, complex, single copy organelle, which serves as the entry site into the secretory pathway. It is crucial for folding and maturation of functional secreted and transmembrane proteins, for lipid biosynthesis and regulation of Ca2+ homeostasis. Little is known about the molecular pathways allowing and regulating the inheritance of this essential and complex organelle in either yeast or mammals. My proposal approaches this basic cellular biology question with the aim of providing a systematic view of the proteins involved in ER inheritance and their functions. I hope to achieve this by combining high-throughput screens followed by the creation of genetic interaction maps and careful genetic and biochemical follow up experiments. By performing this in both yeast and mammalian cells I will be able to compare and contrast this process in both organisms. The information obtained by such an analysis will be immediately relevant to understanding the various human diseases caused by perturbation of ER function. The completion of this project will not only shed light on an unsolved problem in cellular biology. It will also provide a new systematic approach for exploring complex biological processes. With the help of the CDA award, I hope to have the funds as well as the scientific support of the HFSP community that will enable me to tackle this ambitious goal.

For my short-term HFSP fellowship, I propose to develop an in vivo imaging assay to capture the generation of sensory placodal neuroblasts at a high single cell resolution using a long-term chick embryo slice culture system developed by Storey/Swedlow groups at the University of Dundee in Scotland. This would allow the first direct visualization of placode formation in amniotes. I aim to image two dynamic cellular processes: patterns of mitoses underlying neurogenesis in the placodal ectoderm and ingression of placodal cells from the ectoderm. I will develop a long-term cranial slice culture technique for time-lapse imaging in three dimensions using deconvolution microscopy. Using available software created by the Swedlow group and contributing to development of new software for cell tracking, I will employ 3D nuclear tracking and cell membrane tracing to analyze mitosis and cell migration, respectively. For my future directions, I am interested in applying my proposed imaging assay as a tool to elucidate conserved and key molecular and cellular mechanisms underlying cell motility of migratory cell types, such as placode and neural crest, and extend my study to disease tissue models for cancer and fibrosis. I would greatly benefit from an award of the HFSP fellowship to learn and develop imaging techniques and software for image analysis as I move into a new research direction at the onset of my post-doctoral research.

Biological clocks regulate many physiological and cellular functions. Mammalian central circadian oscillator resides in the suprachiasmatic nuclei but circadian clocks also exist within peripheral tissues. Retinal circadian clocks play a critical role in organismal physiology. Molecular regulation of biological clocks involves Clock/Bmal1 heterodimers that activate the transcription of Per and Cry genes. After nuclear accumulation, Per and Cry inhibit their own transcription. Understanding circadian physiology is critical for the comprehension of retinal function either under healthy as well as under pathological conditions.
Sirtuins are histone deacetylases that have pathophysiological relevance to aging, cancer, and metabolism. SIRT1 is expressed in a circadian manner in hepatocytes and fibroblasts and is required for high-level circadian expression of several clock genes. However, little is known whether SIRT1 regulates circadian rhythms in vivo. We would like to hypothesize that SIRT1 regulates circadian rhythms in vivo and we will use retina as a model system to test it.
The ultimate goal is to determine the circadian expression pattern of retinal clock genes in SIRT1 retinal targeted deleted mice. We will: a) determine whether SIRT1 is circadianly expressed in retina, b) specifically delete SIRT1 in the retina to determine if expression patterns of clock genes and proteins are affected, and c) examine whether retinal function is affected in the retina-specific SIRT1 deleted mice.
This short-term fellowship will let me learn about a cutting edge field to bring back this technology and to establish the basis for a novel line of research in my own laboratory in Argentina.

The subventricular zone (SVZ) is a neurogenic niche adjacent to the lateral ventricle that contains a population of stem cells responsible for the generation of olfactory interneurons. SVZ stem cells reside in a specialized microenvironment composed of many different cell types in a milieu rich in extracellular matrix (ECM). It is not known to what extent SVZ stem cell identity is hardwired or modulated by extrinsic niche factors and which niche components regulate neurogenesis. An overlooked component of the SVZ niche is the cerebrospinal fluid (CSF), a product of the Choroid Plexus (CP) that constantly bathes the lateral ventricles. I hypothesize that the CP secretes factors into the CSF that influence stem cell function. I will transcriptionally profile purified CP cell populations and will analyze the components of the CSF by Mass Spectrometry. To verify the function of the identified factors, I will set up in vitro and in vivo validation systems to assess the phenotypic effects of candidates on SVZ stem cell self renewal and differentiation. This study will provide the tools to ultimately target and functionally modulate specific cell populations within the niche. By changing to the neural stem cell field and dissecting the role of the niche in modulating stem cell function I will have an outstanding opportunity to train in neurobiology and learn new theoretical and practical approaches. This will provide an ideal basis to become an independent researcher and study the parallels of niche components in distinct self-renewing tissues.

Schizophrenia is a severe and debilitating mental illness that affects up to 1% of the worlds population, and studies have established there is a strong genetic basis for this disease. Disrupted in Schizophrenia (DISC1) has emerged as one such candidate gene since it was discovered as a chromosomal translocation that segregated with schizophrenia and other mental illnesses in a large pedigree. Furthermore, mice expressing mutations in DISC1 display schizophrenia- and depression-like behaviors, validating human studies implicating DISC1 in mental illness. Recently, the laboratory of Li-Huei Tsai discovered that DISC1 interacts with Wnt signaling to regulate proliferation of neural stem cells (which give rise to neural and glial cell types) in the developing cerebral cortex. Given these results, I hypothesize that mutations in DISC1 give rise to schizophrenia/mental illness phenotypes by disrupting Wnt signaling and neural stem cell proliferation, thereby promoting premature cell genesis and dysfunctional cortex development. To test this hypothesis, I will express DISC1 mutations in neural stem cells in vitro and in vivo to determine their effects on Wnt signaling, proliferation, and the timing or numbers of neural cell types generated. Moreover, since DISC1 is expressed in the adult hippocampus where defects in neurogenesis play a role in major depression, I will ask if DISC1 mutations affect adult neural stem cell function, and if this results in depression-like behaviors. Together these experiments will attempt to elucidate the functional role of DISC1 in neural stem cells which will aid in the development of targeted drug therapies to treat mental illnesses.

Understanding the relationship between membrane protein structure and function is critical both to understanding how cells interact with their environment and to managing disease. Many questions about how membrane proteins fold and oligomerize have not yet been resolved. Model systems that can be used to understand and test principles of interaction or that can help elucidate structure are badly needed. The small multidrug-resistance transporter, EmrE, is a membrane protein that is a promising target for the development of such methods. It has been shown to embody multiple types of interactions, and quick functional tests are available. My proposal begins with the study of the role of particular transmembrane (TM) helices in EmrE in both stabilizing and destabilizing the functional dimer. Then, using the information gathered about the role of each helix, I will seek to develop three different approaches that will be generally applicable to studying membrane protein interactions. 1) An assay to test antiparallel dimerization of two TM helices in the membrane, 2) A method to test designed TM helices and/or create helices that stabilize the oligomerization of two other helices in the membrane 3) A method to map the distance between TM helices in heterodimeric membrane proteins.

Solid tumors comprise a variety of cell types, including neoplastic cells, stromal cells, vasculature and inflammatory cells. The latter contribute to carcinogenesis through multiple mechanisms. Emerging evidence suggests an important role for tumor associated macrophages (TAMs) in tumor growth and metastasis, via its effect on enzyme activity and angiogenesis. However, the initial stages of this involvement, namely, the recruitment of the circulating monocytes to the tumor and their subsequent differentiation to TAMs are currently not fully understood. During my postdoctoral research I intend to address these issues. First, I will isolate the heterotypic signals that initiate monocyte extravasation to the tumor, and will identify subsets of circulating monocytes that respond to these signals. Next, I will examine the molecular pathways that are involved in the differentiation of these cells to TAMs, which is accompanied by arrest and accumulation in hypoxic areas of the tumor. Studying the behavior of TAMs, both in a physiological mouse model, as well as at the molecular level, will assist me in obtaining a comprehensive view on the role of inflammatory processes in malignant tumor progression. The proposed project differs from my previous studies in that I will be addressing issues of cancer and inflammation, in contrast to my current research of hematopoietic stem cell regulation. I anticipate that this change of the research field, combined with the methodologies and experimental strategies that I have gained through my current studies, will equip me with a broad perspective on scientific concepts in current molecular biology and a thorough approach to tumorigenesis.

The precise connectivity of the nervous system is indispensable for its proper function part of this precision is reflected by how neuronal activity controls the exact number of excitatory synapses. The molecular mechanisms linking neuronal activity to synapse-number are still poorly understood, but seem to involve a genetic program including transcription factors of the MEF2-family and one of their target-genes, the E3 ubiquitin ligase Ube3a. Ube3a, mutated in the neurodevelopmental disease Angelmann Syndrome (AS), is enriched postsynaptically and its catalytic activity is necessary for controlling the correct number of excitatory synapses. The aims of my postdoctoral research are to identify the postsynaptic Ube3a-substrates and to elucidate how their Ube3a-mediated ubiquitination and degradation affects the number of excitatory synapses. Specifically, I will identify Ube3a-substrates by a candidate approach and in an unbiased proteomic screen. Localization of candidate-proteins, their interaction with Ube3a and their activity-induced Ube3a-dependent ubiquitination will be tested in hippocampal cultures and sections of wild-type and Ube3a-deficient mice. Identified Ube3a-substrates will be functionally evaluated in organotypic hippocampal slice-cultures by a lentiviral complementation-strategy that knocks down the endogenous protein and expresses an exogenous ubiquitination-resistant mutant morphological and electrophysiological parameters will be assessed. This project will integrate my molecular and cell-biological expertise with biochemical and electrophysiological skills that I will acquire under the supervision of Dr. Michael Greenberg at Harvard Medical School.

The potential use of embryonic stem cells for regenerative medicine has gained high interest over the past years. However, the fact that early human embryos have to be destroyed to obtain such cells has raised many ethical concerns. The recent discovery that four transcription factors, Oct4, Sox2, Klf4 and c-Myc induce pluripotency in mouse fibroblasts in vitro provides a novel source to obtain ES-like cells in a test tube without the need of human eggs. The present proposal aims to further elucidate the molecular mechanisms underlying in vitro reprogramming of somatic cells. I will investigate whether Oct4, Sox2, Klf4 and c-Myc reprogram keratinocytes with the same efficiency than fibroblasts or whether additional proteins are required. Using insertional mutagenesis I provide a strategy to isolate novel candidate genes that are involved in inducing pluripotency in somatic cells. In a second project, I seek to identify chemical compounds that enhance reprogramming of fibroblasts and/or induce pluripotency in the absence of c-Myc. Subsequently, I will perform detailed molecular analyses to elucidate how certain elements in the above proposed research projects induce pluripotency in somatic cells.

Intelligent behavior depends on the ability to generate flexible, context-specific actions. Of special importance for this capacity are the prefrontal cortex, which provides “executive control” and specifies the context or rules currently in effect, and more posterior areas in the frontal and parietal lobe that guide immediate, specific acts. I will examine how these two systems interact, that is, how contextual information in the frontal lobe shapes activity in sensorimotor areas, through combined neurophysiological experiments in awake behaving monkeys and neural network simulations in robotic models. The experiments use an eye movement task - the “antisaccade/prosaccade” task in which monkeys make a saccade (rapid eye movement) either away from a salient cue, or toward the cue based on an earlier instruction. I will examine the functional interactions among three interconnected areas implicated in this task - the dorsolateral prefrontal cortex (dPFC), the lateral intraparietal area (LIP), and the frontal eye field (FEF). I have three specific aims: 1) to characterize the propagation and transformation of top-down signals related to task performance through dPFC, LIP and FEF 2) to probe how top-down signals influence visual and motor responses in LIP and FEF and 3) to evaluate possible algorithms for solving this task using computational modeling in robots. Through combined use of neurophysiological experiments and robotic modeling I hope to provide important new insight into the computations behind the antisaccade task and into the cognitive/sensorimotor interface underlying flexible behavior.

Germinal centers (GCs) are important sites to establish humoral immunity. A cardinal event in GCs is antibody affinity maturation, in which B cells undergo somatic hypermutation of their antibody genes and selection for mutant clones that have acquired increased affinity for antigen. However, the precise mechanism by which selection occurs in GCs remains elusive. An important question is how high- and low-affinity B cells compete for binding antigen and make decisions on their fate. My proposed study is aimed at clarifying this point and eventually the selection mechanism of GC B cells. To accomplish this specific aim, I will study the behaviors of GC B cells in living lymph nodes with a special focus on the competitive aspect of B-cell selection using two-photon laser-scanning microscopy in combination with experimental skills that I have acquired in my previous research. In the first part of this proposal, I will visualize antigen itself utilizing quantum dots, nanometer-size fluorescent particles, to track antigen distribution within GCs and antigen capture by B cells. With this antigen-tracking system, I will analyze the changes in antigen-capturing behaviors of GC B cells and their interactions with follicular dendritic cells and helper T cells during affinity maturation. To directly examine the competition between B cells, I also propose to establish a system in which the fate of high- and low-affinity B cells can be tracked within the same GC. I believe this study would promote comprehensive understanding of the mechanism underlying antibody affinity maturation and shed light on the essence of humoral immunity.

The vertebrate heart is the first organ to form during embryogenesis. The higher vertebrate heart is composed to four chambers separated by septa and connected to two separated out flow tract with spiraling. Each set of chambers serves its specific individual function for maintenance of blood flow throughout the body. Perturbations of this delicate process leads to congenital heart defects (CHDs). Recent remarkable advances in transcription factors (TBX5, TBX1, NKX2-5, GATA4) have resulted in a wealth of information about cardiovascular development and CHDs. However, complicate gene-combinations and epigenetic signal pathway are necessary to determine regional intricacies of each organ development or various phenotypes in CHDs. Chromatin structure is one of the most important regulatory mechanisms that control access of transcription factors to their DNA binding elements and to regulate their target gene expression. Based on our novel concept that Baf60c, a co-factor of SWI/SNF chromatin remodeling factors is essential for cardiac development (Lickert&Takeuchi Nature2004, Takeuchi P.N.A.S.2007), I will investigate three core cardiac chromatin remodeling factors, Brg1 in SWI/SNF, SNF2h in Chrac and Mi-2 in NurD. Thus chromatin-remodeling complexes not only modulate chromatin assembly, but also have specific function with tissue specific factors to establish heart formation. Elucidating these epigenetic function will illuminate greatly our understanding the molecular mechanisms of intricate four-chamber heart and CHDs. Also, identification of their function will provide us new insight of cell specification, cell lineage switching or cell fate in cardiogenesis.

Interactions between stem cells and their surrounding microenvironment, or niche, are critical for the establishment and maintenance of stem cell properties. The adult Drosophila testis contains a discrete morphological stem cell niche, the ‘hub’, that provides an excellent model for the study of niche development. The small cluster of non-dividing, somatic hub cells at the anterior tip of the fly testis is contacted by the germline stem cells (GSCs) that retain their stem cell character through the direct association with the hub. I have demonstrates that integrin-mediated adhesion is important for the correct positioning of embryonic hub cells during gonad morphogenesis and is essential for maintaining the hub cell niche in the adult Drosophila testes. I have developed tools and techniques that allow me to use the hub cells as a model to analyze the mechanisms that underlie stem cell niche morphogenesis. With the HFSP Career Development Award I wil address three aspects of stem cell and niche morphogenesis. First, I propose to identify and analyze genes that control hub cell morphogenesis. Second, I will analyze the morphogenetic events that follow GSC and SSC division to give the first differentiated sperm precursor, the cyst cell encapsulated gonialblast. Third, I will analyze the how the hub controls asymmetric cell divisions in the GSCs. With the help of the HFSP CDA I will establish an indepedent research program that address a fundamental biologlical problem and that will be the at the heart of my research program for years to come.

Cytokines are a family of secreted signaling proteins that regulate immunity, inflammation, and hematopoiesis. Cytokines function by binding to specific receptors on target cells which convey the signal through associated Janus kinases (JAKs) to STAT transcription factors. The cytokine family comprises a broad range of structurally diverse proteins, including interleukins. Interleukin-15 (IL-15), which regulates the development and homeostasis of certain cells of the immune system, signals through a trimeric receptor consisting of an alpha, beta, and a common gamma chain [gamma(c)]. The gamma chain is shared by receptors for other interleukins. Furthermore, IL-15 and IL-2 use the same beta chain in their receptors. Despite the similarity of their receptors, there are distinct differences in the quality and potency of the intracellular responses triggered by these two interleukins. The proposed project aims to elucidate different aspects of interleukin signaling: The interaction of IL-15 with the extracellular domains of its receptor will be analysed to identify the principles that govern the specific interaction between interleukins and their receptors. As IL-15 and IL-2 and the other four gamma(c)-dependent interleukins have only low sequence homology, structural studies will be essential to dissect shared versus ligand-specific binding determinants in interleukin-receptor recognition. Furthermore, the studies of the extracellular components will be extended to the inside of the cell to investigate the interplay between receptors and JAKs. Cell biological, biochemical and biophysical techniques in addition to X-ray crystallography will be employed in this project.

Encoded in the genomes of animals, plants, unicellular green algae, and viruses, microRNAs (miRNAs) are evolutionally conserved among species, and are supposed to act as key regulators of cellular processes. miRNAs usually induce translational repression by binding to partially complementary binding sites in the 3' UTR of their target mRNAs. However, the exact mechanism for the translational repression remains elusive. Many experiments have been reported, but some support the repression at the initiation stage and the others support the repression at the post-initiation stage, making the conclusion controversial. We have established a unique in vitro system that recapitulates the repression at both initiation stage and post-initiation stage. Moreover, our recent findings enabled us to selectively program distinct Argonaute proteins, the core component of the effector complex, the RNA-induced silencing complex (RISC). By combining the in vitro translational repression system and the selective Argonaute programming, we finally have adequate tools for the biochemical dissection of the miRNA-mediated translational repression. We are especially keen on understanding the functional difference between Ago1 and Ago2 in the repression at the initiation stage and the post-initiation stage, and the correlation between repression and deadenylation/degradation of the target mRNAs. Understanding the mechanism of the miRNA pathway will be indispensable for the clinical application of miRNAs. Our study will greatly contribute to that goal by clarifying and consolidating the elusive conclusions preciously reported.

Circadian clocks play an important role in plants, where they regulate a large number of genes in anticipation of changes in light at dawn and dusk. A combination of biochemical modelling and luciferase imaging experiments has resulted in a detailed model of the clock system in Arabidopsis thaliana, consisting of three or more interconnected feedback loops. However, for many purposes it is desirable to work with a simpler model organism, where the clock system is less complex and high-quality measurements are easier to perform. Such an organism is the alga Ostreococcus tauri, the smallest known free-living eukaryote. The aim of this project is to build and refine a detailed model of the circadian clock of Ostreococcus, using data from LUC gene fusion experiments and other sources. I will perform computer simulations of the system as needed to determine the parameter values of the model, and to identify further experiments to be performed. To address questions about the evolution of circadian clocks, I will make less-detailed models that are more amenable to simulated evolution, and with those models investigate the connection between environmental factors and clock design. Finally, more detailed model of Ostreococcus will be examined in a similar context, to identify conditions under which alternative designs of the clock system would be advantageous.

Nuclear reprogramming is the functional conversion of the genome contained within a differentiated somatic cell to a state of developmental pluripotency, through factors that reside in the pluripotent stem cells, such as embryonic stem cells (ESCs). The aim of this research is to decipher the molecular mechanism of reprogramming, through understanding the roles of two factors recently discovered to play essential roles in reprogramming: Polycomb repressive complex 2 (PRC2) and Oct4. Nuclear reprogramming will be studied using cell fusion between mouse ES and human B cells. PRC2 methylates histone H3 and represses many differentiation-specific genes, but also appears to upregulate a set of genes in ESCs. I will modulate expression of target genes from both categories to test their roles in reprogramming. I will also test PRC2’s requirement in reprogramming cell types at different stages of differentiation, in an attempt to decipher the nature of PRC2’s role in reprogramming. Oct4 is a transcription factor that plays a central role in maintaining ESC identity. To identify Oct4 targets that play roles at different stages of reprogramming, I will immunoprecipitate Oct4 at different times after cell fusion. Identified targets will be tested for their essentiality in reprogramming by RNAi. The significance of this proposal is two fold. First, by advancing our understandings in the reprogramming process, we will further our understandings in ESC identity and the nature of each differentiation processes. Second, this work provides essential knowledge for generating patient-specific stem cells used for cell replacement therapy.