Utpal Banerjee - US grants

Neuronal development in the Drosophila eye follows a non-clonal mechanism where cells are instructed by their microenvironment to take on the fate of identified neurons. The sevenless gene participates in this process and mediates the development of the R7 photoreceptor neuron. We have isolated a dominant second site suppressor of the sevenless phenotype. It is called Suppressor of sevenless (Sos). We have mapped this mutation and shown that the suppression is specific to the E4 allele of sevenless, that makes a full sized protein product. We propose that the mutant product of the Sos gene is compensating for the defect in the sevE4 protein, and that in wild type flies, the product of the sevenless gene has a function relevant to the sevenless pathway. Techniques from Drosophila genetics and molecular biology will be used for detailed characterization of the Sos gene. The aspect of sevenless function that the suppressor interacts with will be determined by molecular analysis of the sevenless allele that is suppressed. Genetic mosaic studies will identify the cell(s) responsible for the suppressor function. The Sos gene will be cloned and characterized. From a detailed study of the suppressor, we hope to obtain a better understanding of a pathway that leads to neuronal differentiation.

Preliminary studies on the strawberry notch (sno) gene suggest that it functions together with the genes Notch, Serrate, deltex and the Enhancer of split complex in establishing a pathway of cell-cell communication that leads to the specification of cell fates. The sno mutation not only shares many of the phenotypes associated with mutations in these genes, but also displays strong genetic interactions with them. Additionally, duplications of Notch suppress most of the phenotypes observed in sno flies. A genetic analysis of sno has been completed, and it has been determined that the function of sno is needed in several specific steps of development in many different tissues. The sno gene has been localized through deficiency mapping to a single band of the X- chromosome. As part of this proposal, Dr. Banerjee will isolate the sno gene by chromosomal walking and characterize its gene product. A functional analysis will be initiated to determine the role of sno in the Notch related pathway. He hopes that the characterization of sno will help us understand the molecular basis for permissive interactions that are crucial in the determination of cell fate.

DESCRIPTION (provided by applicant): Cells from a large precursor pool are often chosen to differentiate along specific developmental fates. In many systems, this is achieved through extensive cell-cell interactions utilizing one of many canonical signal transduction mechanisms. The cell's fate is determined, not based on its lineage, but on its immediate environment. The Drosophila eye is an excellent genetic model for understanding the molecular basis for such signaling pathways. Cells interact through the EGFR, Sevenless and Notch signaling pathways to develop into clusters of photoreceptor neurons and non-neuronal cells of the eye. The activation of the proper signaling pathway generates a mosaic of specific transcription factors within the developing field, which then determine the fate of the cells. It is the aim of this application to determine how networks of multifunctional signaling pathways create the differential expression pattern of cell-specific transcription factors. First, genetic and molecular analysis will be used to determine how one signaling pathway could initiate another in a sequential manner. The possibility that the EGFR signal activates the ligand Delta (D1) that then initiates the Notch pathway in the neighboring cell will be investigated. The mechanism by which a single transcription factor can both activate and repress genes in the same cell in response to signals will be determined by studying the negative regulation of the deadpan gene. A gene called runt that is only expressed in two photoreceptor cells in the eye, R7 and R8, will be analyzed to determine how repetitive signaling can provide the context for the expression of the same protein in precisely two cells that develop at some temporal distance. The mechanism by which a set of precursor cells can change fate while retaining their pluripotent nature will be investigated through molecular and genetic analysis of the enhancer of the lozenge gene. Finally, in a large mutant screen involving the generation of mutant clones in the eye, we will search for novel genes of developmental relevance that control the expression of lozenge (lz) and Dl, or control the developmental fate of non-neuronal cells in the eye.

DESCRIPTION (provided by applicant): Drosophila melanogaster has been acknowledged as a premier genetic model system for understanding gene function, developmental networks and molecular basis for genetic disorders including cancers. Drosophila has blood cells or hemocytes that are important for innate immune functions as well as tissue remodeling and wound healing. We initiated a molecular genetic analysis of Drosophila hematopoiesis with the goal to understand the relationship of this process to vertebrate blood development and disorders such as Leukemia. We found that a gene sharing similarity to the Acute Myeloid Leukemia (AML1) protein is essential for the development of 1 of the hemocyte types in Drosophila, called crystal cells. This work led to a lineage diagram for Drosophila hematopoiesis that showed several conserved components. Also the strategies for this development are conserved with similar signaling pathways involved in Drosophila and vertebrate hematopoiesis. More remarkably, concepts such as a common precursor for vascular and blood cells (hemangioblasts) are also conserved. In this proposal, we will first further analyze the functions of the conserved Notch/ PDGF-VEGF receptor/JAK-STAT pathways in Drosophila hematopoieis. We will develop microscopic imaging methods to analyze the hematopoietic process at a single cell level. We will find the molecular basis for the asymmetry that allows hemangioblast divisions to create mixed cell types. And finally, we will analyze novel genes identified from a genetic screen and initiate new genetic screens to identify precursor and possibly stem cell populations and novel proteins that are involved in Drosophila and vertebrate blood cell maturation.

DESCRIPTION (provided by applicant): Cells from a large precursor pool are often chosen to differentiate along specific developmental fates. In many systems, this is achieved through extensive cell-cell interactions utilizing one of many canonical signal transduction mechanisms. The cell's fate is determined, not based on its lineage, but on its immediate environment. The Drosophila eye is an excellent genetic model for understanding the molecular basis for such signaling pathways. Cells interact through the EGFR, Sevenless and Notch signaling pathways to develop into clusters of photoreceptor neurons and non-neuronal cells of the eye. The activation of the proper signaling pathway generates a mosaic of specific transcription factors within the developing field, which then determine the fate of the cells. It is the aim of this application to determine how networks of multifunctional signaling pathways create the differential expression pattern of cell-specific transcription factors. First, genetic and molecular analysis will be used to determine how one signaling pathway could initiate another in a sequential manner. The possibility that the EGFR signal activates the ligand Delta (D1) that then initiates the Notch pathway in the neighboring cell will be investigated. The mechanism by which a single transcription factor can both activate and repress genes in the same cell in response to signals will be determined by studying the negative regulation of the deadpan gene. A gene called runt that is only expressed in two photoreceptor cells in the eye, R7 and R8, will be analyzed to determine how repetitive signaling can provide the context for the expression of the same protein in precisely two cells that develop at some temporal distance. The mechanism by which a set of precursor cells can change fate while retaining their pluripotent nature will be investigated through molecular and genetic analysis of the enhancer of the lozenge gene. Finally, in a large mutant screen involving the generation of mutant clones in the eye, we will search for novel genes of developmental relevance that control the expression of lozenge (lz) and Dl, or control the developmental fate of non-neuronal cells in the eye.

DESCRIPTION (provided by applicant): In this study we will use the Drosophila eye to understand how metabolic stress caused by aberrant mitochondriaI function can affect proliferation. We have shown that mutations in nuclear genes that encode mitochondria! proteins show a block in the cell cycle. For one such mutation, tend, the cell cycle block is due to the activation of a specific pathway including AMPK and p53 leading to the down-regulation of Cyclin E. Although mutant cells show a remarkable reduction in intracellular ATP levels, they do not die, but differentiate and undergo normal morphogenesis. Preliminary data indicates that the mitochondrion affects the cell cycle in different ways, one through the downregulation of the cell cycle activator Cyclin E, while another through the upregulation of a cell cycle inhibitor Dacapo. In this proposal we intend to dissect the pathways that lead from the mitochondrion to the cell cycle. We will use genetic and cell culture studies to determine how Cyclin E is regulated in tend and Dacapo in pdsw. Additionally many more mutants showing a cell cycle defect were isolated in a genetic screen that we will further characterize to identify genes that link the mitochondrion with the cell cycle machinery. The phenomenon being investigated here is novel, and the Drosophila eye can provide important insights into normal mitochondrial function that is important for the well being of a eukaryotic cell. Furthermore, the work has long-term clinical relevance for tumor growth control and in the understanding of inherited diseases, including those of the eye, caused by mitochondrial dysfunction.

DESCRIPTION (provided by applicant): The mammalian inflammatory response is a complex process that involves many cellular components, systemic effects, and involvement of the vascular system, macrophages and other blood cell types. Inflammation occurs in response to injury or infection that are often coupled with one another, which in turn generates proinflammatory signals including cytokines and prostaglandins. Systemic activation of inflammatory response is essential for healing but is also associated with many disorders. Furthermore, the search for new NSAIDs is an important pharmaceutical endeavor. In this proposal, the creation of a simple, genetically amenable, whole invertebrate animal model for inflammatory response is proposed. Preliminary data show that injury to the Drosophila larval epidermis causes a rapid and systemic NF(B-dependent response in macrophages and that these cells increase prostaglandin (PGE2) production. Drosophila genetics will allow for the rapid identification of components regulating these responses. The goal is to delineate the pathway that links the epidermal breach to the activation of the macrophages and the response of the macrophages, including the mechanism for generating prostaglandins. The experimental approach is comprehensive, featuring several analyses of injury- induced events: the mechanism of NF(B activation in macrophages, NF(B- dependent changes in macrophage gene expression, hematopoietic and functional responses of the blood system, and prostaglandin synthesis and signaling. The product resulting from this study will be an in vivo genetic model with which future genetic and drug screening will be feasible with whole animals.

DESCRIPTION Abstract: The control of metabolic pathways and mitochondrial activity and biogenesis during development will be explored using early mouse embryo and the embryonic stem cells derived from them as a model. This proposal is based on the importance of metabolic activity to human health and disorders and the relative lack of data on the mechanisms that control transition between various modes of metabolic activity during phases of development. Nuclear and mitochondrial activities constantly modulate each other, and the relationship between signal transduction pathways commonly studied during development, cancer and stem cell maintenance and metabolic pathways such as oxidative phosphorylation, glycolysis, mitochondrial biosynthesis and nutrient sensing will be explored. The techniques involved will require in vivo tagging of genes that are relevant to metabolism, then studying progression of metabolic gene activity during very early stages of mouse development. This will be followed by isolation of ES cells from these embryos, and through perturbation of various signaling pathways, the effects on their metabolic status will be identified. The overarching goal of this endeavor is to understand the molecular mechanisms underlying the developmental control of a Warburg effect -like transition between oxidative and glycolytic activity;and also to fully understand the nature of cross talk between the nucleus and the mitochondrion during mammalian development. This model is of relevance to studies on human stem cell biology and cancer. Public Health Relevance: This proposal is relevant to public health from many different angles. Metabolic disorders are of great significance and their study comprises the core of this proposal. However, it is the study of the interactions of metabolic pathways with those that have been linked to cancer and developmental defects, including stem cell related studies, is what makes this study particularly important for understanding human health a

Most modern microprocessors support some form of vector operations that allow the same operation to be applied to small vectors of arguments simultaneously. Studies have shown that use of these instructions can improve the performance of many scientific codes by a factor of 2 or more. Unfortunately, the state of the art in autovectorization falls far short of this goal, only achieving improvements of 20-30% on the same codes.

While studies have shown that current autovectorizing compilers do not identify all of the opportunities for vectorization, little is known about why they fail to do so. The PIs plan to evaluate tradeoffs between different compiler optimizations and vectorization in an effort to understand how optimization choices affect opportunities for autovectorization. They will use an extensive set of benchmarks to evaluate these tradeoffs. This research will make it possible to develop better autovectorizing compilers by avoiding optimization choices that interfere with autovectorization. The performance benefits of such compilers will improve the performance of applications ranging from multimedia software to scientific computing.

Project Summary/Abstract Mitochondrial dysfunction and changes in cellular metabolism trigger several clinical conditions including cancer. It is critically important to understand the molecular mechanisms underlying the control of mitochondrial biogenesis and function, and metabolism in general, during normal development and oncogenic transformation. The remarkable conservation in molecular strategy controlling basic cellular processes allows the exploitation of powerful genetic tools developed in the Drosophila eye to study the control of mitochondria and metabolism. The proposed work intends to determine the mechanism by which developmental pathways in the eye primordium regulate mitochondrial structure and function. Also proposed is a study to better understand the mechanism by which a metabolic shift from oxidative phosphorylation to glycolysis takes place when an oncogene is activated. The proposal has three specific aims. In AIM1, the mechanism of control of mitochondria by Lozenge and its mammalian homolog, the oncogene Runx-1 (Acute myeloid leukemia-1/AML-1) will be explored in the context of the growth promoting Yorkie/Scalloped pathway and the steroid hormone receptor, EcR (Ecdysone receptor). In AIM 2, the role of mitochondrial Complex I and two metabolic enzymes that also function as oncogenes, Sdh and Idh, will be studied in the context of oxidative stress response. In AIM 3, the oncogenic influence on cellular metabolism and the molecular mechanism that causes a metabolic shift towards glycolysis will be investigated.

Non Technical Abstract
The primary objective of this project is to acquire a BioSorter (Union Biometrica) for the University of California, Los Angeles (UCLA). The BioSorter is a unique large-particle flow cytometer that rapidly sorts objects based on patterns of fluorescence or fluorescence intensity, including objects that are too large or fragile for standard flow cytometry. The BioSorter enables automated high-throughput sorting of organisms such as free-living and parasitic worms; fly embryos and larvae; and stem cells, other mammalian cells, and cell clusters. The rapid and precise sorting capabilities of the BioSorter enable screening strategies that will greatly expand the scope and impact of diverse research and education programs at UCLA. The BioSorter will be operated as a core facility on the UCLA campus. Research and training programs that will utilize the BioSorter span the fields of parasitology, neurobiology, developmental biology, biochemistry, genetics, toxicology, and stem cell biology.

Technical Abstract
The BioSorter will be used by students of all levels (high school, undergraduate, and graduate) for cutting-edge research and training. Research programs enabled by the BioSorter include: studies of how human-parasitic worms locate hosts to infect; studies of how environmental toxins affect the reproductive system; studies of stem cell development and differentiation; studies of the molecular and cellular basis of molting; studies of natural variation in populations; studies of the role of stem cells in neural circuit function; studies of mammalian germline function; and studies of brain neurochemistry. Education programs enabled by the BioSorter include: introductory and advanced research training courses for undergraduate students using fruit fly genetics and development as a model system, and a summer research program for students from local high schools. We expect at least 135 undergraduate students and 12 high school students to use the BioSorter each year. Use of the BioSorter in research training courses will expose students to cutting-edge technology and greatly enhance their training experience.

Project Summary/Abstract Drawing the fundamental blueprint of tumorigenesis and cancer metabolism is an important and urgent issue for providing proper targets of cancer therapy. Drosophila has long served as a genetic model for many developmental processes, but its usefulness as a cancer research model has only recently been appreciated. The signaling pathways that control glycolysis, tissue growth, cell survival, cell migration, and mechanisms for enhancing oxygen supply to tumors in Drosophila show remarkable conservation with mammalian systems. This study utilizes powerful genetic tools and reagents available for the Drosophila model system to understand how signaling pathways and metabolic enzymes affect tumor behavior. In particular, we will determine the cause and function of aberrant and seemingly non-functional Notch accumulation during tumor formation as well as analyze oxygen supply and metastasis within the malignant tumor to assess how glycolysis, mitochondrial activity, and TCA cycle are involved and influenced. This proposal consists of three major aims. Aim 1 describes experiments aimed at discovering the molecular mechanism of aberrant Notch accumulation and proposed non-canonical function in glycolytic tumors. In Aim 2 we propose to characterize the regulatory factors on metabolic reprogramming during angiogenesis and metastasis. Finally, in Aim 3, we will identify and characterize the non- dual function of a glycolytic enzyme, which has a great potential to be a target of human cancer therapy. The research we propose will lead to critical insights into how the signaling pathways and glycolytic enzymes regulate cancer metabolism and influence tumor progression. The PI will be involved in mentoring and supervision of the work that will involve the training of 2 postdoctoral fellows and 1 graduate student dedicated to this work. Additionally, the laboratory traditionally trains a large number of undergraduates in research. The typical undergraduate spends 2-3 years in the laboratory; postdocs, 5 years and graduate students 5-6 years. The main scientific disciplines our research encompasses developmental biology and genetics. All graduate and postdoctoral researchers involved are highly knowledgeable in these two fields, and the more experienced members of the lab typically pass along this familiarity to newer members.

This RCN-UBE incubator project will begin building a network of STEM educators at 2- and 4-year colleges employing research deconstruction, a pedagogical strategy developed by the investigators to teach undergraduate students the process of scientific inquiry. As research deconstruction requires no laboratory infrastructure, it offers a low-cost, scalable approach that may be accessible to a wide variety of institutions, including community colleges. Engaging students in scientific inquiry is an important strategy for reducing attrition of students from STEM majors, a national problem that threatens the future of the U.S. as a global leader in science and technology. Providing educators with sustainable options for inquiry-based instruction, particularly at 2-year colleges, may help to increase the number and diversity of future STEM professionals.

External assessment and STEM retention data from implementation over a 10-year period at suggest the approach is effective in teaching students the process of science and increasing persistence in STEM. To determine the extent to which these educational benefits can be reproduced at a 2-year campus and identify adaptations necessary for implementation in a community college setting, the investigators will work with partner community colleges to develop a pilot research deconstruction course. Formative assessment will be used to evaluate affective and cognitive learning gains and, in consultation with an advisory team of community college faculty, identify necessary adaptations for a second course offering. These findings and those of network participants implementing research deconstruction at research universities will be shared at a workshop to identify best practices. To increase awareness of the pedagogy, local STEM faculty will be invited to the workshop, and video recordings of the presentations will be posted on a website for distribution to the broader education community. Outcomes from this incubator will inform future efforts as the network is expanded to include more 2- and 4-year colleges.

This project is being jointly funded by the Directorate for Biological Sciences (BIO), Division of Biological Infrastructure, and the Directorate for Education and Human Resources (EHR), Division of Undergraduate Education as part of their efforts to address the challenges posed in Vision and Change in Undergraduate Biology Education: A Call to Action (http://visionandchange.org/finalreport/).