Cunming Duan - US grants

9728911 Duan The proposed project involves studying the molecular mechanisms by which insulin-like growth factors (IGFs), peptide growth factors/hormones which are essential for normal growth and development, act to control embryonic tissue formation and differentiation in vivo. Recent studies utilizing cultured mammalian cells suggest that the bioactivity of IGFs could be modulated by specific, high-affinity IGF-binding proteins (IGFBPs) in vitro. The in vivo role of each IGFBP in controlling the availability of IGFs in specific embryonic tissues has not been determined. Research in this area has relied heavily on rodent models, and attempts have been hampered by the inaccessibility of the mammalian fetus which is enclosed in the uterus. In this project, Dr. Duan proposes to utilize a model teleost fish, the zebrafish (Danio rerio) to test the hypothesis that the access of IGFs to specific embryonic tissues is regulated by specific IGFBPs expressed locally and that this regulation is crucial for normal tissue growth and differentiation. The accessible, transparent and fast-developing embryos and the availability of numerous genetic mutants make zebrafish particularly suited for investigating the developmental role of IGFs/IGFBPs and the underlying mechanisms. The specific aims of the proposal are to: 1) determine the structure and biological activities of zebrafish IGFBP-3; 2) determine the developmental expression patterns of IGFBP-3 together with IGFs and the IGF-1 receptor; 3) determine the role of IGFBP-3 in controlling IGF actions in developing zebrafish embryos. The expected results should provide direct evidence for the in vivo role of IGFBP-3 in controlling IGF actions in a vertebrate embryo and will further our understanding of how the IGF system acts to control growth and development in vertebrates. Determination of the structures of fish IGFBP-3 and development of the corresponding protein, cDNA probe and antiserum will make available, for the first time, valuable tools for investigation of IGFBP actions in a teleost fish. Information gained from these studies may prove to be valuable to aquaculture for efficient production of animal protein to meet the needs of a continually growing human population.

DESCRIPTION (Verbatim from the application): Injury of the arterial wall results in accelerated vascular smooth muscle cell (SMC) proliferation and directed migration from the media to the intima, leading to intimal hyperplasia and thickening. This process contributes significantly to the pathogenesis of atherosclerosis and related complications. The molecular mechanisms underlying the abnormal SMC proliferation and migration in this process are not well defined but are key to understanding the basis of the development of atherosclerotic lesions. Insulin-like growth factor-I (IGF-1) is a potent regulator of SMC proliferation and directed migration. The growth-promoting and chemotactic actions of IGF-1 are mediated through the IGF-1 receptor (IGF-1R), but how the activation of the same receptor by the same ligand leads to distinct growth and chemotactic responses is unknown. Recently we have discovered that IGF-1 utilizes distinct signaling pathways to stimulate SMC growth and migration. Our further studies indicate that SMCs secrete several high-affinity IGF binding proteins (IGFBPs) and that these IGFBPs may play a critical role in determining whether SMCs will migrate and/or proliferate in response to IGF-1. Our goal in this proposal is to understand how IGF-1, IGF-1R, and IGFBPs interact with each other to regulate SMC proliferation and migration. The first aim in this proposal is to elucidate the divergent growth and chemotactic signaling pathways activated by ligand occupancy of IGF-1R in SMCs. The second aim focuses on the distinct roles of different IGFBPs in determining SMC response to IGF-1. Site-directed mutations will also be generated to determine the specific structural motifs that are essential for the distinct biological actions of different IGFBPs. In the third aim, we will determine the role of cell-surface-associated IGFBP5 in promoting SMC migration towards an IGF-1 gradient. Furthermore, we will test the hypothesis that IGFBP-5 is translocated into SMC nuclei and that the nuclear IGFBP-5 alters SMC motility through a novel mechanism that is independent of the IGF-1R-mediated signaling pathways. The proposed studies will lead us towards an understanding of the molecular interactions between IGF-1, IGF-1R, and IGFBPs and will provide novel information on the regulation of SMC proliferation and migration, as well as a conceptual model of the molecular mechanisms that determine the specific physiological outcomes of IGF-1 stimulation. It is our belief that a complete elucidation of the mechanisms of IGF-1, IGF-1R and IGFBP actions in SMCs should have many ramifications including the development of future therapeutic strategies that may correct or circumvent atherosclerosis and related complications.

The insulin-like growth factor (IGF) signaling system is essential for normal growth and development in vertebrates. The proposed project involves studying the molecular mechanisms by which the IGF signaling system acts to control cell proliferation, differentiation and cell death (apoptosis) in developing vertebrate embryos in vivo. There are three components to the vertebrate IGF signaling system: the IGF ligands, IGF receptors, and IGF binding proteins (IGFBPs). While it is clear that the IGF ligands and receptors transduce signals that positively regulate growth, the specific role of each of the IGFBPs and their interactions with the IGF ligands and receptors in vivo is not well understood. Our laboratory has been utilizing a model teleost fish, the zebrafish (Danio rerio) to investigate the IGFs/IGFBP interactions and their functional importance. We use zebrafish because of their accessible, transparent and fast-developing embryos. In addition, numerous genetic mutants are available for our studies. We have characterized the IGF signaling system in zebrafish and shown that IGFBP-2 inhibits IGF actions in developing zebrafish embryos. Our further studies have revealed the presence of several other IGFBPs in zebrafish. These IGFBPs show different structural characteristics and distinct expression patterns. The goal of this project is to understand how each of these IGFBPs interact with IGF ligands and receptors to regulate cell proliferation, differentiation, and apoptosis in developing zebrafish embryos. The overall hypothesis to be tested is that different IGFBPs are differentially regulated, and they each play a distinct role in specifying the IGF actions in defined embryonic tissues. The specific aims of the project are: 1) to determine the actions of IGFBP-2 in regulating cell proliferation, differentiation, and apoptosis in vivo by targeted expression of IGFBP-2 in the developing eyes; 2) to determine the structure of zebrafish IGFBP-1 and IGFBP-3, produce the corresponding proteins, and analyze these fish IGFBPs biochemically and functionally; 3) to determine the spatial and temporal expression patterns of these fish IGFBPs; 4) to determine the specific effects of IGFBP-1 and IGFBP-3 in controlling IGF actions in vivo by targeted expression in the developing eyes; and 5) to elucidate the physiological functions of the endogenous IGFBPs in vivo using a novel targeted gene "knockdown" approach. The expected results should provide novel insights for the roles of various IGFBPs in controlling IGF actions in a vertebrate embryo and will further our understanding of how the IGF signaling system acts in vivo to control growth and development in fish specifically and in vertebrates generally. Determination of the structures of fish IGFBPs and development of the corresponding protein, cDNA probe and antisera will make available valuable tools for investigation of IGFBP actions in fish. A comparison of the structure and function of IGFBPs from different vertebrate species will provide novel insight to our understanding of the evolution of this important gene family. Information gained from these studies may prove to be valuable to aquaculture for efficient production of animal protein to meet the needs of a continually growing human population.

[unreadable] DESCRIPTION (provided by applicant): Abnormal vascular smooth muscle cell (SMC) accumulation in the intima plays a key role in the pathogenesis of atherosclerotic lesions. Locally produced insulin-like growth factors (IGFs) are important regulators of intimal SMC accumulation. IGFs stimulate SMC migration, proliferation, differentiation, and survival. These diverse actions of IGFs are mediated through the IGF-I receptor (IGF-IR), a transmembrane tyrosine kinase. How activation of the same IGF-IR by the same ligands leads to these diverse biological responses is not well understood, but is key to understanding the molecular basis of the role of the IGF signaling system in development of atherosclerotic lesions. Recently, we and others have identified several high-affinity IGF-binding proteins (IGFBPs) that are synthesized and secreted by SMCs. Our studies indicate that these IGFBPs are important determinants of specific cellular responses to IGF stimulation, and that a key player in this paradigm is IGFBP-5. IGFBP-5 binds to IGF and modulates IGF actions. IGFBP-5 also stimulates SMC migration through a ligand-independent mechanism. Our recent studies reveal that IGFBP-5 is localized in the SMC nucleus and that nuclear IGFBP-5 is likely to be derived from the secreted protein. Moreover, the conserved IGFBP-5 N-domain possesses transcriptional activation activity which is not affected by IGF binding. The overall goal of this proposal is to further elucidate the IGFBP-5 nuclear translocation pathway and to determine its role in regulating SMC migration, proliferation, differentiation, and apoptosis. The first aim will determine the membrane and cytoplasmic proteins that act as key IGFBP-5 partners and mediate IGFBP-5 internalization and nuclear translocation. The second aim will investigate the functional significance of IGFBP-5, with special focus on its nuclear targeting and ligand binding. Native and mutant IGFBP-5 will be expressed in IGFBP-5 siRNA knockdown SMCs and in IGFBP-5 null cells for in vitro studies. Transgenic mice with targeted overexpression of native or mutant IGFBP-5 in SMCs and IGFBP-5 knock-out mice will be used for in vivo studies. The third aim will determine the regulatory mechanism(s) of the transactivation activity of IGFBP-5 and to identify IGFBP-5 target genes. The proposed studies will lead us towards a better understanding of the molecular interactions between IGFs, IGF-IR and IGFBPs and provide novel information on the regulation of SMC migration, proliferation, differentiation, and apoptosis, as well as, a model of the molecular mechanisms of IGFBP-5 actions. It is our belief that elucidating the mechanisms of IGF and IGFBP-5 actions in SMCs will have important applications, including the development of future therapeutic strategies that may correct or circumvent atherosclerosis and related complications. [unreadable] [unreadable]

Insulin-like growth factors (IGFs) are a family of peptide growth factors/hormones that are essential for normal animal growth and development. Recent studies suggest that the availability and bioactivity of IGFs are regulated by several specific, high-affinity IGF-binding proteins (IGFBPs). IGFBPs belong to a conserved gene family of proteins that bind to IGFs in extracellular environments. Since the binding affinities of IGFBPs for IGFs are equal to or even greater than those of the IGF receptors, they can control the amount of IGFs presented to their cell surface receptors and may play key roles in determining the biological activities of the IGF signaling pathway. There is also in vitro evidence that some IGFBPs have intrinsic biological activities that are IGF-independent. This project is designed to test the hypothesis that different members of the IGFBP family are expressed in spatially and temporally restricted fashions and they each play distinct roles in regulating tissue growth and differentiation through IGF-dependent and -independent mechanisms. The current project has 3 aims. In the first aim, the mechanistic basis of IGFBP-3 action in regulating head skeleton and inner ear growth and differentiation will be investigated with special focus on the role of ligand binding and nuclear targeting. For this, native and mutant IGFBP-3 will be expressed in IGFBP-3 knocked down embryos for in vivo mechanistic studies. Aim 2 seeks to determine the structure of zebrafish IGFBP-4, map its spatial and temporal expression pattern, and investigate its physiological functions in vivo. In Aim 3, the mode of IGFBP-4 action and the possible role of proteolysis in controlling local IGFBP-4 action will be studied in vitro and in vivo. Aim 4 seeks to determine zebrafish IGFBP-6 structure, developmental expression, physiological functions in vivo, and elucidate the molecular mechanisms underlying its actions. The completion of the proposed studies will provide novel and important insights into the functions of IGFBPs and the mechanistic basis underlying their biological actions. For the first time, the functional significance of ligand-independent actions, nuclear localization, and proteolysis of an IGFBP will be examined in vivo. An understanding of the underlying mechanisms of growth regulation in fish will undoubtedly contribute to vertebrate growth physiology in general. A better understanding of the underlying mechanisms of growth regulation in zebrafish should help to understand human growth physiology and may have applications in aquaculture for efficient production of animal protein as well. This research project is also of importance for understanding the structural and functional evolution of the IGFBP gene family and will expand research infrastructure in Michigan and increase research and training opportunities for undergraduate and graduate students.

This workshop titled "Zebrafish- a Model System for Exchange of Ideas, Integration of Knowledge and Collaboration between Developmental Biologists and Comparative Endocrinologists" will be held as a special event coordinated with the 6th International Symposium on Fish Endocrinology in Calgary, Alberta, Canada. The workshop will mainly focus on the functions and molecular mechanisms of hormones and their receptors and will be divided into three sessions: 1) Zebrafish-A Model for Comparative Endocrinology Session; 2) Zebrafish-A Model for Developmental Endocrinology Session; and 3) General Discussion. The goals of this workshop are to promote the exchange of information and international collaborations between developmental biologists and comparative endocrinologists, inform investigators about the genomic and molecular tools available in the zebrafish model, and ultimately facilitate the growth of the field of "developmental endocrinology". Scientists of different nationalities, genders, and career stages will participate in the workshop. Travel awards for graduate students, especially those from underrepresented groups, will be provided. It is anticipated that this workshop will advance discoveries, not only in the basic sciences, but also help develop applications in related fields such as biotechnology and aquaculture.

Two international scientific meetings will be supported at the University of Michigan in July, 2011: the inaugural meeting of the North American Society for Comparative Endocrinology (NASCE 2011), and the 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology (ISAREN 2011). The NASCE is a new scientific society formed in 2010 with the purpose of promoting the comparative study of hormones and hormone action. This includes topics in evolutionary, environmental (including endocrine disrupters), general and biomedical endocrinology/neuroendocrinology. The ISAREN was formed in 1992 to promote the study of the endocrinology and neurobiology of amphibians and reptiles.

The NASCE 2011 and ISAREN 2011 meetings will revitalize and strengthen the field of comparative endocrinology in North America by bringing together students, young investigators, and trainees from diverse areas and backgrounds to exchange ideas and to establish and strengthen collaborations. We promote diversity in our field of science as well as diversity of scientific topics in the meeting program, and we provide significant opportunities for groups traditionally underrepresented in science to attend the meeting. Meeting abstracts will be published in the open access journal Frontiers in Endocrinology, and the plenary lectures and selected symposium presentations will be published in the journal General and Comparative Endocrinology. This will allow for the presentations to be widely disseminated to the scientific community.

The fundamental mechanisms underlying the aging process are poorly understood. This project will employ a unique vertebrate model animal to conduct molecular, physiological, and genetic experiments to determine the role of insulin/Insulin-like growth factor (IGF) signaling in aging. The hypothesis to be tested is that IGF signaling exerts pro-aging and/or anti-aging effects depending on the stage of life, tissue, and presence of different IGF binding proteins. The information gained from these studies will provide novel insights into the mechanisms by which the IGF pathway regulates aging. While this work pertains specifically to fish, it will have broad relevance in understanding the nature and evolution of aging regulation in general. A better understanding of the nature and evolution of aging regulation will likely lead to the development of diagnostic and therapeutic tools that will increase the healthy lifespan of the aging population in the United States. The project will also yield new information about growth, nutrition, and aging interactions and may have applications in aquaculture for efficient production of animal protein. This project will integrate research with education by directly incorporating the knowledge gained from these studies into undergraduate courses. The PI teaches Animal Physiology and Molecular Endocrinology. The proposed studies are directly relevant to the topics covered in these two undergraduate courses. This project will provide valuable research and training opportunities for young scientists and students. The graduate and undergraduate students supported by this grant will participate in every aspect of the cutting-edge research. Results will be broadly disseminated. These studies will benefit society as a whole through interactions with the public by various outreach programs.

DESCRIPTION (provided by applicant): Animals with experimentally manipulable genomes have been invaluable for aging research. In model organisms such as worms, flies, and mice, the ability to specifically add or delete a gene enables the characterization of the roles of specific gene(s) in lifespan regulation. While these animal models have contributed, and will continue to contribute, to our understanding of aging biology, there are gaps in the range of experimental strengths provided by each of the existing animal models. This project will focus on a new and promising vertebrate model organism, the annual turquoise killifish (Nothobranchius furzeri), which has a lifespan of just several months. N. furzeri provides many additional advantages, such as the ability to produce many sibling progeny from a single mating, low cost of maintenance, amenability to forward genetic screens, and the ability of their eggs to be stored dry at low temperatures for as long as a year. Despite these advantages, the N. furzeri model has not been widely used in the field of aging research. This is in part because genetic manipulation experiments were not possible in this species. Recently, my lab has succeeded in developing methods for generating stable and inducible transgenesis in this species. Drawing on our research experience with fish biology and the IGF signaling pathway, the goal of this application is to develop new genetic tools for conducting loss-of-function studie and to use them to test the hypothesis that insulin-like growth factor binding protein-3 (IGFBP-3), a major IGFBP, regulates lifespan via IGF-dependent and/or -independent mechanisms. IGFBP-3 regulates IGF availability by binding IGF tightly and releasing it only under certain conditions. IGFBP-3 also has IGF-independent actions. It is possible that this conserved and multifunctional protein may play key roles in modulating aging. In Aim 1, we will develop genetic tools and use them to determine the role of IGFBP-3 in lifespan regulation. Aim 2 will investigate the functional significance of ligand-binding and nuclear localization of IGFBP-3 by generating and studying inducible transgenic lines that express either wild type or a mutant form of IGFBP-3. The completion of the proposed studies will provide novel insights into the roles of IGFBP-3 in the longevity of vertebrates. The anticipated results will also lead to the development of much needed methods and genetic tools to knockout a gene of interest or to induce gene expression in a tissue-specific and temporally restricted manner in this emerging vertebrate model. The methods and transgenic tools developed in this project will be broadly disseminated and distributed to the scientific community. These genetic tools and methods, combined with the unique features and the extremely short-lifespan of N. furzeri, will open many new avenues for investigations and should facilitate new discoveries in vertebrate aging biology. A better understanding of the biology of aging regulation will likely lead to the development of diagnostic and therapeutic tools that will increase the healthy lifespan of the aging population in the United States.

How newborn neurons are organized into the right locations with the right timing to form functional circuits is a fundamental question in developmental neurobiology. An attractive model system in which to address this question is the Gonadotropin-releasing hormone (GnRH) neuronal system. GnRH neurons are master regulators of the reproductive system. They secrete GnRH peptides to initiate reproductive activity. Abnormal development of GnRH neurons can lead to reproductive disorders. Although significant advances have been made in understanding the central importance of GnRH neurons in reproduction, very little is known about where the GnRH progenitor (i.e., undifferentiated) cells are located in the developing nervous system and how they are instructed to become GnRH neurons. This project fills this gap in our knowledge. The anticipated results will provide new knowledge on how insulin-like growth factor (IGF) signaling, a major embryonic growth-promoting pathway, regulates GnRH neuronal development. The knowledge gained from this project may have applications in the aquaculture industry and will help understand the etiology of reproductive disorders. This project provides training opportunities for high school, undergraduate, and graduate students, and postdoctoral fellows. The students participate in all aspects of this cutting-edge research project. In collaboration with the University of Michigan Museum of Natural History, the project also includes the development of a summer camp curriculum in neurobiology, entitled "Shining Neurons in the Brain!", for K-12 students. This and other outreach activities make this NSF-funded research accessible to the public.

An attractive model system in which to address the question of how newborn neurons are organized into the right locations with the right timing to form functional circuits is the GnRH neuronal system. GnRH neurons emerge from the nasal/olfactory placode in early embryos and undertake a long-distance migration from the olfactory region to the preoptic area and hypothalamus. The embryonic origin(s) of GnRH progenitor cells is still under debate. Little is known about how these progenitor cells are specified to give rise to GnRH neurons. Most vertebrate species have a second GnRH gene that is expressed in the midbrain. Much less is known about the GnRH2 neuron development and regulation. This project tests the hypothesis that the two types of GnRH neurons originate from the cranial neural crest- and/or optical placode-derived progenitor cells and that IGF signaling regulates the emergence of GnRH neurons at the right time and location by controlling the proliferation, migration, and/or survival of their progenitor cells. Perturbation of IGF signaling in a critical time window during early embryogenesis alters the temporal and spatial organization of the newborn GnRH neurons, which, in turn, affects reproductive function later in life. Aim 1 investigates the embryonic origins of GnRH neurons; Aim 2 determines whether IGF signaling regulates the emergence of the GnRH neurons by controlling migration, proliferation, and/or survival of their progenitor cells, and Aim 3 determines the long-term reproductive consequences of this regulation.