Anne E. Griep - US grants

DESCRIPTION (provided by applicant): Maintaining proper control over cell growth and differentiation is fundamentally important not only during embryonic development but also throughout the life of the animal when tissue homeostasis and self-renewal are required for long-term survival. Despite their importance, the molecular mechanisms through which these cellular processes are controlled in vivo are only poorly understood. The ocular lens has become an important model system for evaluating the roles of specific gene products in regulating growth and differentiation in vivo. In recent years, we and others, through the analysis of transgenic and mutant mice generated by gene targeting strategies, determined that the retinoblastoma susceptibility protein, i.e., is an essential regulator of cell cycle withdrawal during lens fiber cell differentiation. More recently, we also learned that the pRB-related proteins, p107 and/or p130, play a role with pRB in cell cycle control both in the undifferentiated epithelium and during fiber cell differentiation. Although it is thought that the E2F transcription factors are critical targets of pRB proteins during lens fiber differentiation, the specific roles, if any, for individual E2Fs in mediating normal growth and differentiation or the effects of Rb inactivation, remain to be determined. Factors other than the pRB family members contribute to the regulation of cell growth and differentiation in the lens. Our recent studies suggest a heretofore unrecognized role of the PDZ domain proteins, discs-large (DLG), a tumor suppressor in Drosophila, in regulating cell proliferation and structural integrity in the epithelium and newly differentiating fiber cells. In this renewal application, we propose four aims to further our understanding of the roles of these factors in regulating lens cell growth and differentiation in vivo. The aims of the proposal are to: (1) determine if PDZ domain proteins are required for regulating cell cycle and epithelial cell structure in the lens epithelium; (2) determine the role of pRB and pRB-like proteins in cell cycle control in the undifferentiated and differentiating cells of the lens; (3) determine the role of pocket protein-E2F interactions in cell cycle control during fiber differentiation; (4) determine if pRB and/or pRB-like proteins play a role as a differentiation factor in lens differentiation. Together, the results of these studies will contribute to our understanding of how tumor suppressor proteins regulate cell proliferation and differentiation in vivo and how disrupting their function contributes to human disease such as cataracts.

The Transgenic Animal Facility provides UW Comprehensive Cancer Center investigators with the capacity to manipulate the genome of experimental animals used in cancer research. The efficient generation of knockout of transgenic animals requires specialized technical skills in the areas of animal husbandry and surgery, embryology, embryonic stem culture and gene targeting vectorology, embryo micro-manipulation, and cryopreservation. In addition, the procedures require sensitive and expensive microscopic equipment as well as SPF-animal facilities. Because most individual investigators cannot meet these requirements within their own laboratories, gene targeting and transgenic animal technologies must be provided by common resource facilities. At the University of Wisconsin, that resource is the Transgenic Animal Facility (TAF). The cancer research performed at UWCCC relies heavily upon this resource. For that reason, funds are requested to support this critical activity.

DESCRIPTION (provided by applicant): Regulation of lens development requires the coordinated activities of a number of growth factor signaling pathways, cell-cell and cell-matrix adhesion complexes, and cell cycle regulators. An important question is how all these different pathways are spatially and temporally coordinated so as to ensure proper lens formation. PDZ (PSD95/DLG/ZO-1) proteins are proteins that have the potential capacity to link all of these molecular pathways. Through their capacity to act as scaffolds, they assemble large protein complexes that, depending on the constituent molecules assembled, signal towards different cellular fates. In Drosophila, the tumor suppressor genes Discs Large (dig) and Scribble (scrib), which encode two PDZ proteins, are essential for establishing and maintaining normal epithelial cell structure, polarity and proliferation. Our recent transgenic mouse studies using of the E6 oncoprotein from human papillomavirus as a dominant inhibitor of PDZ proteins, suggests a heretofore unrecognized role for PDZ proteins such as Dlg-1 and Scrib in many stages of lens development. We also have shown that a hypomorphic allele of Dlg-1 gives rise to cataractous abnormalities in the mouse lens during embryogenesis. In this application, we propose to use a combination of genetic and molecular analyses on mouse mutants in Dlg-1, Scrib, and other interacting genes to (1) determine the requirements for Dlg-1 and/or Scrib in lens development, (2) determine if, and the mechanisms through which, Dlg-1 and/or Scrib regulate lens development by modulating cell adhesion protein complexes, and (3) define the pathway through which Dlg-1 and/or Scrib regulate the cell cycle. Loss of cell cycle control, cell architecture and polarity are observed in many cataractous conditions ranging from congenital cataracts to age-related cataracts in adults to secondary cataracts, that occur following cataract surgery. Factors that regulate these fundamental properties of lens cells during embryogenesis have the potential to be relevant to maintaining normal lens structure and transparency throughout the life of the animal. Additionally, defects in PDZ and polarity genes recently have been found to be associated with retinal degenerations in experimental animals and humans. Thus, the knowledge we gain from our novel studies to understand the role of Dlg-1 and Scrib in mouse lens development potentially will have significant impact on our understanding not only of lens development but also cataract, and other ocular diseases.

Transgenic and genetically-engineered mutant animals provide one of the most valuable mechanisms for generating in vivo models for unraveling the molecular and genetic mechanisms of cancer and for evaluating new strategies for treatment and prevention studies. Efficient generation of transgenic animals requires great technical skill that is acquired only after extensive experience with animal husbandry and breeding, animal surgery, embryo manipulation and micromanipulation. The transgenic Animal Shared Service makes transgenic technology accessible and affordable to the members.of the University of Wisconsin Comprehensive Cancer Center. It uses the personnel, facilities and equipment of the campus-wide University of Wisconsin Biotechnology Center's Transgenic Animal Facility. Services currently provided by the Transgenic Animals Core include: Generation of transgenic mice and rats by pronuclear microinjection Cryopreservation of mouse and rat embryos [unreadable] Generation of knockout mice: generation of targeting vectors, generation of targeted embryonic stem (ES) cell clones, and generation of chimeric mice by injection of ES cells into host blastocyst Strain rederivations and ovarian transplants Other miscellaneous molecular biology and embryo services related to transgenic and knockout mouse production Consulting and training In addition, the staff are available for consulting at any point in the process of using the services. Considerable time is spent discussing experimental design and providing guidance on processes that they themselves are or will "be carrying out. They also train investigators in techniques such as superovulating, mating, and collecting reproductive tracts from female mice for embryo cryopreservation, isolating DMA for pronuclear micronjections, mouse handling and breeding protocols, and cutting tails and genomic DNA preparation Southern blotting to identify correctly mutated ES cells.

The overall goal of this Program Project grant application is to elucidate the mechanisms of autoimmunity to type V collagen in heart and lung transplants. The use of animal models to study mechanism is essential for advancing our understanding of this important clinical problem. To this end, the focus of the Transgenic Mouse Core (Core B) is to provide the technical expertise for the generation of genetically modified mouse strains and to maintain a colony of these unique mouse strains such that mice are generated for the experiments that address the aims of the proposed three Projects. To do so, the first part of Core B will utilize personnel in the University of Wisconsin Biotechnology Center's (UWBC) Transgenic Animal Facility (TAF) to (1) carry out gene targeting experiments in embryonic stem cells to produce ES cell clones with genetically altered alleles and the blastocyst microinjections to generate new mouse mutant strains carrying these mutant alleles, (2) to rederive genetically modified strains that are obtained from other institutions or are housed presently in non-SPF animal facilities so that the mouse stocks for this project can be housed in a newly opened specific pathogen free vivarium at the University of Wisconsin, and (3) cryopreserve for long term storage and safe keeping all the genetically modified mouse strains used in this proposal. In the second part of Core B, Core staff will (1) verify the genetic identity of all the mutant mouse strains rederived for the projects and the inducible tissue specific expression of certain strains, (2) maintain a breeding colony of all the genetically modified mouse strains such that the required numbers of the mouse stocks can be supplied to the individual Projects as needed for their experimentation. This Core will provide specific services for each of the three projects in this proposal. The services provided by this Core will be essential for achieving the overall goals of this grant application.

PROJECT SUMMARY / ABSTRACT Genetically engineered mice and rats are widely used as models for defining the molecular mechanisms underlying cancer etiology and for evaluating new strategies for cancer treatment and prevention. The Genome Editing and Animal Models Shared Resource (GEAM), formerly the Transgenic and Mutant Animal Facility, has served as a shared resource for the University of Wisconsin Carbone Cancer Center (UWCCC) for over two decades and is one of the elite facilities within the United States in offering a comprehensive array of services related to generation and preservation of genome-edited animal models. The primary mission of the GEAM is to make state-of-the-art genome editing technologies accessible to UWCCC members. Specific Aim 1 is to provide the expertise and infrastructure required to generate novel and relevant genome-edited or transgenic animal models and genome-edited cell models for use in cancer research. GEAM staff are capable of serving UWCCC members in all aspects of experiment planning and execution, including design of efficient and specific approaches to achieve the desired loss, gain, or alteration of gene function using CRISPR/Cas9 or other genome editing and transgene-based approaches; design, production and use of the required genome editing reagents or transgene vectors; identification of animals that carry the desired genome edit or transgene; and minimization of off target edits. Our skills in reproductive biology, embryo manipulation, and animal husbandry enable us to edit the genomes of inbred mouse and rat strains that exhibit low reproductive capacity. Specific Aim 2 is to provide state-of-the-art services that allow valuable animal models to be banked and recovered as needed to preserve these animal models or reduce the costs associated with maintaining live breeding stock for novel models that are not actively being studied. GEAM staff are highly experienced and capable of cryopreserving mouse and rat embryos or sperm and recovering mouse and rat models through embryo transfer or in vitro fertilization. In addition, we are capable of rederiving mouse and rat strains to eliminate pathogens that may compromise research or prevent animal models from being imported into our vivaria or shared between investigators working at different institutions. Since its inception, GEAM has generated hundreds of transgenic, knockout, and genome-edited mouse and rat models for UWCCC members. Sixty-one unique UWCCC members have been served during the current CCSG funding cycle, an increase of 30% compared to the previous grant cycle. Support from the CCSG allows our services to be provided to UWCCC members at costs far below those of commercial vendors or similar cores at other research universities. Convenient access to our first rate, cost-effective services enhances the ability of UWCCC investigators to conduct innovative research that advances the UWCCC strategic mission.

PROJECT SUMMARY/ABSTRACT The primary function of the lens is to focus light on the retina so as to allow us to see. This function relies on the transparency of the lens, and this transparency depends on the precise organization of its primary constituent cell type, the lens fiber cells. Our goal is to understand what determines the correct cellular organization within the lens that confers its transparency. As lens fiber cells differentiate, they elongate and make direct interactions between themselves to create a highly ordered, tissue-wide cellular organization that confers the overall architecture of the tissue and its transparency. Any disruption of this organization leads to opaque lenses (i.e. cataracts) that are the most common cause of blindness world-wide. We recently discovered two proteins that dictate lens architecture: they are Dlg-1 and Scrib1. Furthermore, we determined that Dlg-1 is critical for conferring planar cell polarity (PCP) in mice. PCP refers to the coordination of cells such that they move, orient, and/or proliferate in a particular direction critical for the normal morphogenesis of the relevant tissue. Another core PCP protein, Vangl2, has been implicated in determining lens shape. Here we provide evidence that Scrib as well as the newly identified PCP protein, Dlg-1, and the well-established PCP protein, Vangl2 determine overall lens architecture. They do so by determining the overall shape of lens fiber cells, and the cell:cell contacts between lens fiber cells within the equatorial region of the lens. Unclear is whether this is due to PCP-dependent or PCP-independent effects of these proteins on lens architecture. The primary goal of this exploratory R21 grant application is to distinguish between these two possibilities. We will approach this using genetic approaches that have been well-validated in other invertebrates as well as more detailed mechanistic studies including the use of state-of-the-art high resolution STED microscopy to resolve the interactions between core PCP proteins and other factors involved in determining lens shape. The two aims of this R21 proposal are to 1) Determine whether planar cell polarity is key to determining lens architecture and 2) Define the interplay between planar cell polarity factors and cell:cell adhesion factors in determining lens architecture. Completion of these studies will provide important and novel insights into the mechanisms required to achieve and maintain lens transparency, a research priority of the NEI, and have an impact on the overarching NEI goal of better preventing and treating cataracts.