Grant R. MacGregor - US grants

Defects in the regulation of apoptosis are associated with a variety of human disease including AIDS, cancer and neurodegeneration. BCL-2 is the founder member of an expanding family of related gene products that control apoptosis during human embryonic development and adult homeostasis. However, the number of genes involved, the developmental processes that they regulate and the extent to which they overlap in function are not well understood. We have identified a new member of the BCL-2 gene family named Bcl-w by random insertional mutagenesis in the mouse. Mice lacking Bcl-w display a variable growth deficit and a severe testicular atrophy. The testis phenotype involves an arrest in germ cell development followed by a loss of both germ and Sertoli cells. A molecular analysis indicates that BCL-w is closely related to Bcl-2 and is expressed in similar embryonic and adult tissues. We hypothesize that Bcl-w mediates cell survival during embryogenesis and adult homeostasis by regulating apoptosis in discrete tissues. To test this hypothesis, we propose four groups of experiments. First we will characterize the expression pattern of BCL-w during mouse embryonic development and in adult tissues. Second, we will identify essential functions for Bcl-w during development by analyzing the extent of programmed cell death in embryos and adult mice lacking Bcl-w. Third, we will study the function of Bcl-w in spermatogenesis by determining (a) if Bcl-w is required in a cell autonomous manner for haploid germ cell development and (b) whether interaction with the extracellular matrix influences Sertoli cell survival in the absence of BCL-w. Fourth, we will determine if Bcl-2 compensates for loss of function of BCL-w by analyzing animals that lack both Bcl-w and Bcl-2. The results will provide novel insight regarding the function of this new member of the Bcl-2 gene family in mammalian development. Such information may ultimately be used to help develop molecular therapies of human disease that arise from deregulated apoptosis.

[unreadable] DESCRIPTION (provided by applicant): Each year, over 1 million abortions are performed in the US as a consequence of unintended pregnancy. Unintended pregnancies therefore have a significant negative impact on both the individual and society. Contraception is an effective method of reducing unintended pregnancies. One way to further reduce the rate of unintended pregnancies is to develop safe and effective methods for reversible male contraception. Hormone based approaches have been developed for use in men. However, these can have significant side effects including the length of time required for both contraception and its reversal. In theory, a specific inhibitor of spermiogenesis could be a powerful contraceptive method, as it should take effect, and be reversed, relatively quickly. Thus, a challenge for basic research is to improve our understanding of the gene products and pathways required for spermiogenesis so that some of these discoveries may be translated into novel approaches for male contraception. Towards this goal, we have used random insertional mutagenesis in mice to identify genes required specifically for mammalian spermatogenesis. Homozygous symplastic spermatids (sys) male mice are sterile due to a defect in spermatid-Sertoli cell adhesion just prior to spermatid elongation. The sys mutation involves a deletion of 1.2 Mb of mouse chromosome 14 that appears to contain only one, novel gene. This gene is predicted to encode a membrane-anchored structural protein with a proline rich N-terminus. We hypothesize that a defect in this novel gene is responsible for the sterility in sys homozygote males, that this gene functions in the Sertoli cell to mediate spermatid-Sertoli adhesion, and that inhibition of the genes function in fertile adult male mice could block spermatid development. Five specific aims are proposed to test this hypothesis. First, we shall generate male mice with a specific mutation of the novel gene and verify that they are sterile. Second, we shall use germ cell transplantation to investigate whether the function of the novel gene is required in germ cells or somatic cells within the testes. Third, we shall analyze the expression pattern and subcellular distribution of the gene product in testes using RNA in situ hybridization, immunohistochemistry and biochemical methods. Fourth, we shall investigate how the gene product functions by identifying proteins that interact with it in the testes. Finally, we shall investigate whether inactivation of this gene in adult fertile male mice can block spermiogenesis. The results of this basic research will provide information about a new gene product whose function is required for spermatid-Sertoli adhesion. They will also provide an assessment of the suitability of this novel gene product as a potential target for development of new methods of male contraception. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Human cardiovascular and pulmonary dysfunction is often life threatening and generates a huge economic burden. Such conditions can arise from abnormal development, or post-natal, from infection, acute injury or exposure to environmental allergen. The high incidence of these conditions provides a clear rationale for long- term basic research to understand the molecular basis for normal development and homeostasis of cardiovascular and pulmonary systems. The long-term goal of this research is to understand the function of the novel protein fibronectin type-III domain containing 3B (FNDC3B) in mammalian cardiovascular and pulmonary development and homeostasis. Fndc3b is expressed in cardiovascular and pulmonary systems and mice mutant for Fndc3b display respiratory distress and die shortly after birth. The cardiovascular system of Fndc3b mice is malformed and hearts display ventricular septal defects and non-compaction. Lung development is also affected and the conducting airway epithelium has abnormal morphology. FNDC3 proteins contain a highly conserved consensus binding site for WW-domain containing proteins. Based on these, and additional, findings we hypothesize that FNDC3B functions in pulmonary epithelial cells, and in vascular endothelial cells to modulate cell-cell adhesion required for normal pulmonary and cardiovascular function. We also hypothesize that FNDC3B is required for secretion of surfactant and that FNDC3B accomplishes each of these functions by binding to specific WW-domain containing proteins. To test these hypotheses, three sets of experiments are proposed. First, we will identify which cells express FNDC3B in the lung and heart of mice. Second, we will analyze the anatomy and function of epithelial cells lining the bronchiolar airway and alveoli and investigate whether the expression of proteins required for normal pulmonary function is affected in Fndc3b mutant and control animals. We shall also investigate whether cell- cell junctions are intact, and if unique secretory organelles required for normal function of airway epithelial cells, atrial myocytes and vascular endothelial cells such as lamellar bodies, secretory granules, caveolae and Weibel-Palade bodies are abnormal in Fndc3b mutant animals. Third, we will investigate which WW-proteins interact with FNDC3B using (a) an array based method and (b) by tandem-affinity purification of epitope- tagged FNDC3B expressed in mouse airway epithelial MEL15 cells and HUVEC endothelial cells followed by mass spectrometry and database analysis. These studies will provide new information about the function of FNDC3B in cardiovascular and pulmonary development in mice. The long-term benefit of this basic research will be to improve our knowledge of the molecular and cellular basis for cardiovascular and pulmonary development and homeostasis, which in turn will offer new opportunities for diagnosis, and ultimately treatment, of human conditions associated with dysfunction of these essential systems. PUBLIC HEALTH RELEVANCE: Abnormal cardiovascular and lung function in newborn infants is a common cause of morbidity and mortality that generates significant human suffering, and financial burden on the health-care system. Understanding the causes of these disorders is an important step towards the long- term goals of improved diagnosis and treatment for such conditions. We are investigating the function of a novel protein, FNDC3B, which is required for normal heart and lung development and post-natal survival of mice. The long-term benefit of this basic research will be to increase our knowledge of the molecular and cellular basis for cardio-pulmonary development in mammals, which in turn will offer improved diagnosis, and ultimately treatment, of human conditions associated with abnormal cardiovascular and pulmonary function.

Core 003 - Basic - Project Summary/Abstract - Transgenic Mouse Facility (TMF) The Transgenic Mouse Facility (TMF) shared resource communicates awareness about existing and emerging methods and resources involving genetically modified mice to Cancer Center investigators and helps them incorporate these tools into their research programs. The TMF provides services on a recharge basis that require specialized training, are technically difficult to perform, or require expensive equipment. The services include design, development, re-derivation, cryopreservation, and re-animation of genetically modified mice in an efficient and cost-effective manner. The TMF website (http://research.uci.edu/tmf/index.htm) lists information about new resources from the literature as well as available services including (a) consultation on strategies to modify the mouse genome, (b) insertion of conventional multi-copy transgenes and bacterial artificial chromosomes (BAC) at random loci via pronuclear injection of DNA, (c) targeted insertion of single- copy transgenes at the ROSA26 locus via homologous recombination in ES cells, (d) targeted mutagenesis of endogenous loci in ES cells, (e) targeted mutagenesis of loci in mice via Cas-9, (f) cryopreservation of mouse sperm and preimplantation-stage embryos, (g) importation, export, rederivation or reanimation of mouse strains via IVF or embryo transfer, (h) breeding and genotyping of mouse strains, (i) Southern blot analysis of targeted loci in ES cells and animals. The TMF also collaborates with CFCCC members to develop or import new methods involving genetically modified mice at no initial cost to the investigator. Consultation regarding projects continues to be provided at no cost. In addition to CFCCC Members, the TMF supports investigators at many different universities and corporations within the USA. These include academic institutions (UCLA, UCSC, UCSB, UCR, UCSF, Stanford, USC, VA) and corporations (Irvine Scientific, Isis Pharmaceuticals, Peregrine Pharmaceuticals) in CA. The TMF also services investigators at institutions in 17 additional states, including 7 other NCI-designated CCCs: UCLA- Jonsson; UCSF - Diller; OHSU - Knight; MUSC - Hollings; Stanford Cancer Institute; St. Jude's Children's Research Center, USC-Norris. A link on the TMF home page invites investigators to contact the TMF Managing Director and Scientific Director to ask about resources for their use, including advice on mouse models of cancer, identifying and importing strains of mice with cancer susceptibility, and mouse genetics resources of use in studying the cancer problem. TMF personnel subsequently identify resources to meet the researcher's needs and provide these at the lowest possible cost.

ABSTRACT The goal of the Disease Model Development and Phenotyping Project (DMDPP) is to produce and characterize the next generation of animal models for AD that accurately model the pathology of late onset AD (LOAD) and to provide predictive models for therapeutic development. These models will be generated under transparent and open intellectual property conditions. The Jackson Laboratory will conduct 2nd-site validation of LOAD models and will ensure their broad availability and rapid dissemination to all researchers. The foundation of this endeavor is our innovative APP knock-in (APP-KI) mouse that expresses humanized Aß at physiological levels, and which exhibits amyloid plaque deposition (see introduction). We will complete our base platform for development of mouse models of LOAD, by humanizing the mouse Tau (Mapt) locus by replacing coding exons of mouse Tau with those from the human Tau (MAPT) locus. To double homozygous APP-KI and hTau (APP-KI+/+/hTau+/+) mice, we will add the major risk factor for LOAD, APOE4. To expedite analysis of additional factors influencing LOAD, the DMDPP will make extensive use of CRISPR/Cas9 technology to generate animal models that express polymorphisms in risk factors identified from genome wide association studies (GWAS), including TREM2, PICALM, BIN1, CD2AP, ABCA7 and EPHA1. The effect of each GWAS allele in generating a LOAD phenotype will be determined when combined with APP-KI, and hTau. Subsequently we will combine specific GWAS alleles to investigate for synergistic effects on LOAD pathology. The incorporation of multiple GWAS polymorphisms on the APP-KI+/+/hTau+/+ background and production of cohorts of mice for analysis will be accelerated using IVF and embryo transfer instead of standard breeding. The molecular pathological phenotypes in these LOAD models will be characterized at an unprecedented level of detail through a novel and innovative immunological approach, using conformation dependent and aggregation specific monoclonal antibodies that distinguish eight different types of amyloid deposits in humans and transgenic mice. The molecular phenotype will also be comprehensively determined by quantifying neurons and microglia, synaptic loss, soluble and insoluble tau and Aß and markers of phospho tau. The core will also characterize the gene expression profile of the models by RNAseq and epigenetic markers, as well as structural, functional, and diffusion magnetic resonance imaging. The behavioral phenotype will be characterized by elevated maze, open field, and novel object recognition. The extensive data generated will be used to compare the molecular pathology to that of LOAD and to decide which lines to advance for further development and testing to produce the next generation of animal models.