Bruce H. Appel - US grants

DESCRIPTION (Adapted from the applicant's abstract): Proper nervous system function requires that neural tissue be precisely patterned so that different kinds of neurons and glia develop at correct times and places and axons reach their appropriate targets. In vertebrate embryos, floorplate has a critical role in patterning, as it is the source of molecules that influence neural cell fate and guide axons. Thus, understanding how floorplate cells are specified is fundamental to understanding neural development and function. The research proposed uses genetic analysis to test the role of Delta-Notch mediated cell interactions and learn how multiple gene functions are integrated as a means to elucidate the mechanisms that specify cell fates in the vertebrate midline. Specifically, this application first proposes to learn how fates of individual precursor cells correlate with their positions in the midline, as well as how neighboring precursor cells interact during gastrulation. Experiments are then proposed to learn how expression of genes important to midline development corresponds with the distribution of precursor cells in the midline. Next, it addresses the role of Delta-Notch mediated cell interactions in patterning the midline through observation of cell behaviors in mutant embryos and disruption of Delta-Notch interactions specifically at the midline. Finally, it proposes to combine mutant analysis and gene overexpression experiments to investigate how multiple signaling pathways are integrated to pattern the midline and specify floorplate.

? DESCRIPTION (provided by applicant): The long-term goal of this project is to understand how oligodendrocytes, the myelinating glial cells of the central nervous system (CNS), are produced. During development, dividing neuroepithelial precursors give rise to oligodendrocyte progenitor cells (OPCs). Following migration to become dispersed throughout the CNS, some OPCs differentiate as myelinating oligodendrocytes but others remain in a non-myelinating state into adulthood. The mechanisms that guide formation of OPCs from proliferating neuroepithelial precursors and specify myelinating and non-myelinating fates are not well known. Using zebrafish as a model system, this project combines in vivo live cell imaging with genetic and pharmacological manipulations to test novel hypotheses formulated to identify mechanisms that regulate OPC specification. Specific Aim 1 will test the hypothesis that microRNA inhibition of apical Par polarity proteins regulates the proliferation to differentiation transition by making precursors less sensitive to Shh signaling. Specific Aim 2 will test the hypothesis that different levels of Shh signaling specify myelinating and non-myelinating OPCs. Specific Aim 3 will identify gene functions that mediate specification of myelinating and non-myelinating OPC fates in response to differential Shh signaling. Completion of these Aims will fill important gaps in our basic knowledge of neural development and reveal molecular targets that could be used to promote myelination following injury or disease.

[unreadable] DESCRIPTION (provided by applicant): The goal of this project is to develop zebrafish as a model system to investigate regeneration of oligodendrocytes, the myelinating cell type of the vertebrate central nervous system, from resident populations of neural stem and progenitor cells. The ability to promote formation of new oligodendrocytes might aid therapies to restore nervous system function following disease or injury. Zebrafish offer numerous advantages for studying neural regeneration. Zebrafish develop entirely outside the mother and embryos and young fish are optically clear, providing the opportunity for direct and extended observations of neural cells. To facilitate direct observation of oligodendrocytes, we have produced transgenic reporter gene lines that express fluorescent proteins in oligodendrocytes at all stages of their differentiation. Because they readily take up chemical compounds, our transgenic zebrafish can be used to screen for drugs that promote oligodendrocyte replacement. Thus, this work seeks to utilize the Zebrafish Chemical Screening Center to search for drugs that promote formation of oligodendrocytes. Neural diseases and injuries have devastating impacts on human health. This work will provide a foundation for identifying and investigating genes and drugs that promote neural regeneration and recovery. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The term goal of this project is to produce a comprehensive and detailed understanding of the cellular and molecular mechanisms that guide formation of peripheral nerves in vertebrates. During development, numerous connections are established between the central nervous system (CNS) and the body via peripheral nerves. These nerves are responsible for conveying information in two directions. Some nerves carry signals out of the CNS to the body to control movement and autonomic functions whereas other nerves transmit information about external stimuli from the body to the CNS. Failure to properly form or maintain peripheral nerves results in a large array of peripheral neuropathies that are manifested as, for example, muscle weakness and loss of muscle control, gastrointestinal dysfunction, inability to sense pain or change in temperature and neuropathic pain. Our approach to investigating peripheral nerve development is to create transgenic zebrafish that express fluorescent proteins in different cellular components of peripheral nerves and then combine in vivo, time-lapse imaging with tests of gene function. In preliminary studies we determined that perineurial cells, which form a protective sheath around myelinated motor nerves, originate as glial cells in the ventral spinal cord and that they are essential to motor nerve development. In Specific Aim 1 of this project we will investigate the molecular mechanisms that specify ventral spinal cord precursors to develop as perineurial cells. In Specific Aim 2 we will investigate the roles of perineurial glia and Schwann cells in motor nerve formation. In Specific Aim 3 we will characterize mutations that disrupt Schwann cell development and myelination as a way to identify genes that are necessary for motor nerve formation. Completion of these aims will greatly extend our understanding of both cellular behavior and gene function in peripheral nerve development, facilitating effective design of therapy intended to treat peripheral neuropathies. PUBLIC HEALTH RELEVANCE: Failure to properly form or maintain peripheral nerves results in a large array of peripheral neuropathies that are manifested as, for example, muscle weakness and loss of muscle control, gastrointestinal dysfunction, inability to sense pain or change in temperature and neuropathic pain. This project will identify cellular behaviors and genes that are necessary to peripheral nerve development.

DESCRIPTION (provided by applicant): The long-term goal of this project is to identify the genes that are required to build a nervous system. The nervous system is composed of two general classes of cells: neurons, which transmit information in the form of electrical impulses, and glial cells, which facilitate impulse transmission and survival of neurons. How neurons and glial cells are formed and organized into functional neural circuits during development is still poorly understood. The project design draws from the well-validated idea that mutations that disrupt neural development reveal critical genes. Building on the expertise of the collaborating team of investigators, this work will discover genes that are necessary for development of motor axons and glial cells that wrap central and peripheral nerves. The project will use zebrafish as a model system, which permits the careful observations necessary to find rare mutations that cause specific neural defects. The first aim of the project is to screen families of mutagenized zebrafish embryos for defects of motor axons and two distinct populations of central and peripheral myelinating glial cells by examining expression of transgenically encoded fluorescent reporter proteins that reveal cell morphologies. The second aim is to determine how each mutation causes its corresponding defect through careful characterization of cellular defects, using immunohistochemical and transgenic cell marking techniques and in vivo time-lapse imaging. The third aim is to determine the chromosomal location of each mutation and identify the affected genes. Completion of this project will enhance understanding of the genetic bases of neurological disorders involving the sensory system or glia and provide genes that might be used to promote neural repair following injury or disease. PUBLIC HEALTH RELEVANCE: This project will identify genes that are necessary to build a nervous system. It will provide a better understanding of how the nervous system develops and potentially lead to new strategies to promote neural repair following disease or injury.

? DESCRIPTION (provided by applicant): The long term goal of this project is to understand how oligodendrocytes, which are glial cells of the central nervous system, ensheath specific axons with specialized, proteolipid-rich myelin membrane. To myelinate axons, oligodendrocytes extend numerous membrane processes than spirally wrap axons. However, not all axons are myelinated. Based on observations that brain activity can modify myelin, we hypothesize that activity-dependent signals from axons influence which axons are selected for myelination. Using zebrafish as a model system, this project combines in vivo live cell imaging with genetic and pharmacological manipulations to investigate mechanisms that guide formation of myelin on specific axons in response to neuronal activity. Specific Aim 1 will test the hypothesis that neuronal activity provides axons with a competitive advantage for myelination through direct observation of ensheathment and myelination of identifiable axons that have different electrical activities in vivo. Specific Aim 2 will use genetic and pharmacological approaches and live imaging to test the hypothesis that neuronal activity promotes myelin sheath growth on select axons by activating the PI3K-Akt-mTor signaling pathway. Specific Aim 3 will test a hypothesis that activity-mediated signaling promotes the stability and translation of mRNAs that encode myelin proteins and identify new activity- regulated transcripts. Completion of these aims will substantially extend our understanding of the cellular mechanisms by which oligodendrocytes choose axons for myelination and key molecules that promote activity- regulated myelin membrane growth. The results of this project have the potential for important new insights to learning, memory and psychiatric disease and to provide a foundation for designing therapeutic strategies to promote myelination of brains damaged by disease or injury.

Project Summary How axons are selectively ensheathed by specific amounts of myelin is a fundamental and unsolved problem in neurobiology. Myelin, produced by oligodendrocytes in the central nervous system of vertebrate animals, enhances axonal conduction of electrical impulses and provides axons with metabolic support. Evidence accumulated over the past several years indicates that myelination can adapt to neuronal activity, and that adaptive myelination is essential for learning and new skill development. The next important step is to identify the molecular functions that mediate communication between neurons and oligodendrocytes to facilitate adaptive myelination. Building on our own evidence that activity-evoked axonal vesicular secretion promotes myelin sheath growth, we hypothesize that proteins that function in synaptic communication between neurons also mediate synaptic-like signaling between axons and oligodendrocytes. We will test this hypothesis using zebrafish and mice, building on our expertise in zebrafish genome modification, mouse in utero electroporation and imaging of oligodendrocyte lineage cells in both species. Our experimental plan has two parts. First, we will investigate the expression and subcellular localization of synaptic proteins identified by RNA-seq profiling of oligodendrocyte lineage cell transcripts. Second, we will initiate loss-of-function studies to learn if synaptic proteins promote myelin sheath growth in vivo. This project has the potential to reveal important new mechanistic insights to adaptive myelination and the results will create a foundation for future, detailed investigations of axon- oligodendrocyte communication.

PROJECT SUMMARY The long-term goal of this research program is to understand how specific axons of the developing central nervous system (CNS) are ensheathed with specific amounts of myelin, a specialized, proteolipid-rich membrane produced by oligodendroglia. During development, subpopulations of neural progenitors produce oligodendrocyte precursor cells (OPCs), which migrate and divide to populate the CNS. Subsequently, some OPCs differentiate as myelinating oligodendrocytes whereas other OPCs persist through adulthood. Importantly, myelin is plastic and can be modified by brain activity. Recent evidence indicates that changes in OPC proliferation, oligodendrocyte differentiation, myelin sheath characteristics and axon selection for myelination contribute to myelin plasticity. However, the molecular mechanisms that regulate myelination, particularly in response to neuronal activity, are poorly understood. The investigations that comprise this research program focus on three broad areas of developmental myelination. First, using single cell RNA-seq data we generated, we will investigate how neural progenitors are specified as OPCs. Second, will axo-glial interactions and mRNA localization promote myelin sheath growth. Third, we will investigate how microglia, the resident innate immune cells of the CNS, modify myelin coverage on axons in response to neuronal activity. We use zebrafish as a model system, which enables us to combine time-lapse imaging with genetic and pharmacological manipulations to observe and test cell behaviors and myelination in an intact, living animal. The results of this research program have the potential to provide important new insights to the developmental basis of learning, memory and psychiatric disease and to provide a foundation for designing therapeutic strategies to promote myelination of brains damaged by disease or injury.

PROJECT SUMMARY / ABSTRACT The advent of genome editing and induced pluripotent stem cell technologies now provide hope for creating therapies to treat and cure many developmental diseases. However, incomplete knowledge of the genetic regulatory mechanisms that guide cell differentiation and function stands as a significant barrier to progress. Overcoming this barrier will require a workforce with a training foundation that integrates a strong base in genetics, a deep knowledge of developmental, cellular and molecular mechanisms, and the ability to apply basic concepts to the translational potential of stem cell biology. The Genetics of Development, Disease, and Regeneration Training Program seeks to foster a broad foundation of interdisciplinary knowledge and career development that empowers a diverse population of students to apply their training to the study and treatment of developmental diseases. To achieve this goal, the GDDR Program is grounded in five training fundamentals: 1) knowledge base, 2) research skills and innovative technology, 3) hypothesis-driven research, 4) communication skills and 5) professional and career development. The GDDR Program will create a unique training environment by combining the broad and interdisciplinary research interests of our faculty with a training plan that integrates genetic principles and mechanisms into the study of development and stem cell biology. Thirty well-funded and accomplished Training Faculty drawn from 10 basic science and clinical departments create a highly collaborative and interdisciplinary environment. The breadth of our training environment will provide trainees with a diverse menu of labs for thesis training, while maintaining a supportive community and facilitating beneficial interactions between the GDDR Program and other T32-funded programs at the University of Colorado Anschutz Medical Campus. We will take full advantage of these interactions to develop courses and training activities that involve multiple programs. Thus we will broaden the exposure of our trainees to different knowledge, technologies, and career opportunities, and extend the impact of our training program to benefit a larger population of students and researchers at CU AMC. Based on the quality and size of our student pool and Training Faculty, we propose to support six trainees per year, which will permit us to be highly selective while building an interactive group of trainees. Our trainee recruitment plans are structured to attract highly talented students from diverse backgrounds, thereby contributing to a rich setting for training and accomplishment.