Peter J. Donovan - US grants

DESCRIPTION: (Adapted from the applicant's abstract) Formation of fertile oocytes requires orderly segregation of chromosomes during meiosis, which depends on proper assembly of a bipolar spindle and its actions. The PI has identified a novel kinase IAK1, one of very few components identified so far in the female meiotic spindle. The proposed study is to test the hypothesis that IAK1 is an important player of the meiotic chromosome segregation machinery. Specific Aim 1 is to localize the IAK1 protein and kinase activity in female germ cells throughout meiosis. In addition, IAK1 expression patterns and activity will be examined in mouse mutants defective in either checkpoint pathways or MPF/MAPK signal transduction pathways. Specific Aim 2 is to disrupt the function of IAK1 by injection of antisense oligonucleotides, kinase-negative mutant form of IAK1, and anti-IAK1 antibodies into oocytes. Spindle formation and phosphorylation as well as chromosome segregation will be assessed in the treated oocytes. Specific Aim 3 is to determine the functional domains of the IAK1 kinase by introducing wild-type and mutant forms of IAK1 into maturing oocytes. The mutated IAK1 proteins will be taqged with FLAG octapeptides or green fluorescent protein and localized in the injected oocytes by confocal microscopy. Simultaneously, the kinase activity will be examined in the oocyte extracts. Specific Aim 4 is to identify the IAK1-interacting proteins in meiotic germ cells. Immunoprecipitation and Western blotting will examine interaction of IAK1 with known IAK1-interacting proteins in other systems, such as Cdc37 and p100. Novel IAK1-interacting proteins will be identified in yeast two-hybrid screening system using a mouse oocyte/egg cDNA library.

DESCRIPTION (provided by applicant):Primordial germ cells (PGCs) are the embryonic precursors of the gametes of the adult animal. They can give rise to pluripotent stem cells termed embryonal carcinoma (EC) cells during the formation of testicular tumors. PGCs can also give rise to pluripotent stem cells termed embryonal germ (EG) cells when cultured in the presence of three polypeptide growth factors. The conversion of a PGC to a stem cell in vitro occurs in a matter of days. However, the alterations in gene expression that occur in PGCs during this period are unknown. This proposal is designed to test the feasibility of using suppression subtractive hybridization to identify the molecular changes that occur during the transition from a PGC to a pluripotent stem cell using mice as a model system. Identifying the molecular changes that occur during this process will generate new information about the mechanisms controlling i) PGC differentiation during gametogenesis, ii) the processes involved in PGC transformation and iii) developmental potency in mammals. The knowledge of these processes will aid in our understanding of the factors controlling fertility in mammals.

DESCRIPTION: (Adapted from the applicant's abstract) In all vertebrates, oocyte development is arrested at the prophase of meiosis I (MI). Oocytes can remain arrested in this state for the entire reproductive lifespan of the organism; in humans as long as forty years. Resumption of meiosis requires activation of maturation promoting factor (MPF), the complex of cyclin B and cyclin dependent kinase-1 (CDK1/p34cdc2). The mechanism of MPF activation in mammals has remained elusive and the inability to mature human oocytes is a major problem for in vitro fertilization (IVF) clinics. We recently demonstrated that oocytes from mice lacking an MPF activator, the Cdc25B phosphatase, fail to resume meiosis after prophase arrest. Mice lacking Cdc25B provide the first genetic model for prophase arrest in mammals and provide a unique reagent with which to dissect the molecular mechanisms regulating mammalian meiosis. The specific aims of the proposal are designed to answer fundamental questions about the control of meiosis in mammals and provide the scientific foundation for future clinical advances. The specific aims are designed: i) To determine the subcellular localization of Cdc25B in the maturing oocyte, ii) To define the functional domains of the Cdc25B molecule, iii) To determine whether pathways that regulate Cdc25B activity during mitosis also act during meiosis, and iv) To use the Cdc25B protein as a unique tag to dissect the pathway controlling prophase arrest and meiotic resumption in mammals.

DESCRIPTION (provided by applicant): Correct development of Primordial Germ Cells (PGCs), the embryonic precursors of the gametes, is a prerequisite for adult fertility. Failure of PGCs to survive, proliferate or differentiate correctly in the embryo and fetus can result in sterility in the adult animal. In some cases normal PGC differentiation is perturbed and PGCs form pluripotent stem cells, termed Embryonal Carcinoma (EC) cells. These in turn form testicular tumors, teratomas or teratocarcinomas, which are the most common form of cancer in young men. Little is known about the molecular mechanisms responsible for guiding normal PGC differentiation and which are perturbed during testicular carcinogenesis. In vitro, PGCs cultured in the presence of Kit ligand (KL) and leukemia inhibitory factor (LIF) proliferate as long as they would in vivo. But when basic Fibroblast growth factor (bFGF or FGF2) is added, PGCs continue to proliferate and give rise to pluripotent stem cells termed Embryonic Germ (EG) cells. This process mimics the formation of testicular tumors in vivo in which PGCs give rise to pluripotent EC cells. Although EC and EG cells are pluripotent, our preliminary data show that PGCs cannot form any other cell type and are therefore considered nullipotent. Thus the conversion of a PGC into an EC or EG cell represents a conversion from a nullipotent to a pluripotent state. The ability to manipulate PGC potency in vitro with bFGF provides a unique system with which to study the control of developmental potency in mammals. We designed a novel retroviral gene transfer system to dissect and manipulate the signaling pathways activated in PGCs by KL, LIF and bFGF to determine the relative importance of these pathways in stem cell development. Understanding the molecular mechanisms controlling the conversion of a PGC into a pluripotent stem cell will fill gaps in our knowledge of developmental potency regulation, testicular tumor formation and germline development as well as generating general information about stem cell physiology. The Specific Aims of the proposal are: i) To determine the role of bFGF in altering developmental potential of PGCs, ii) To define the mode of action of bFGF in stem cell development, iii) To define the role of bFGF in pluripotent stem cell formation in vivo and iv) To identify genes up-regulated in PGCs following exposure to bFGF.

DESCRIPTION (provided by applicant): Summary Despite the great potential of human embryonic stem cells (hESCs) for advancing new treatments for human disease, there are important unknowns in understanding the stem cell state. Addressing these unknowns could improve the ability to establish pluripotent stem cells: how to maintain them and how to turn them into specific cells. The proposed research strategy will address questions about pluripotency through study of the Wnt pathway effectors, T cell factor and Lymphoid enhancer factors (TCF/LEFs). One member, TCF3, is a transcription factor that in mouse ESCs (mESCs) represses expression of master pluripotency factors Oct4, Sox2, and Nanog (OSN). Importantly, mouse Tcf3 limits pro-pluripotency feedforward expression of these genes during mESC differentiation. While studies with mESC have been useful and show how Tcf3 should be considered the fourth core stem cell regulatory factor, extrapolating the role to hESCs has inbuilt limitations due to key signaling differences between mESCs and hESCs. But TCF3 function in human ESCs is entirely unknown. Our preliminary data show that TCF3 is dynamically expressed in hESCs. By comparison with other core factors, TCF3 protein levels decline rapidly upon differentiation before other changes are seen and may therefore be a critical, first event. TCF3 expression is highly varied in hESC colonies indicating that hESCs may be poised to differentiate. Differentiation is accompanied by rapid upregulation of other TCF/LEFs suggesting switching of Wnt target gene promoter occupancy. The overarching hypothesis is that hESCs express TCF3 which inhibits Wnt signaling, maintains a stem cell chromatin structure and maintains Activin signaling (a key pathway regulating hESC pluripotency), to hold hESCs in a poised state; pluripotent but ready to go. With differentiation signals TCF3 is replaced by activating TCF/LEFs that alter chromatin structure, driving gene expression and cellular differentiation. Specific Aim 1 will test the role of TCF3 in regulating Activin signaling. We will study control of Activin inhibitors, Follistatin and Inhibin E, identified as potential TCF3 target in preliminary studies. Specific Aim 2 will test the role of TCF3 in regulating the chromatin remodeling factor SMARCA2. These studies could define the relevance of TCF3 downregulation for hESC pluripotency and differentiation and place it in the larger hESC regulatory network. Relationships between TCF3 and master regulators (OSN) will also be defined using microarray and ChIP-Seq. Specific Aim 3 will test the role of TCF3 relatives in hESC differentiation. We find hESC differentiation is accompanied by rapid up-regulation of TCF3 relatives, (LEF-1/TCF-1/TCF-4). Overexpression of these factors will be used to test their role in driving hESC differentiation. Using ChIPseq we will test how TCF/LEFs occupy gene promoters to control genes in cell type- specific ways. This research could better characterize pluripotency molecularly and establish how signaling pathways can instigate fate decisions by rapidly altering transcription factor activity. These studies could provide new insights into mechanisms regulating early human development.

PROJECT SUMMARY Stem cell medicine promises to revolutionize the treatment of human diseases and injuries, and has captured the hopes of the scientific community and the public alike. Perhaps nowhere is the potential of stem cells to treat human disease and injury more promising than for neurologic disorders. Traveling a path from ?bench to bedside? is still a relatively new opportunity for researchers and provides novel challenges for training graduate students. Historically, pre-doctoral neuroscience students were trained in understanding the basic biological mechanisms underlying how the brain functions and guides behavior, learning and memory, movement, and to identify the processes that become dysregulated and result in neurological disease. Indeed, the biological mechanisms and causes for many neurological diseases have been identified, paving the way for the generation of transgenic animals and model systems to study neurological disorders. Critically, these advances, combined with the advent of stem cell biology, open a path to treat neurologic diseases with transplantation of stem cell derived cell populations or to develop treatment strategies in stem-cell derived cell models. This is a path that requires training beyond basic science. Accordingly, the goal of our Training Program in Stem Cell Translational Medicine for Neurological Disorders is to provide opportunities for pre-doctoral trainees to perform translational bench research that could treat human neurological disease and injury, in the spirit of the NIH vision for translational medicine. Additionally however, to meet the challenges presented by the advent of this new frontier, we provide opportunities for trainees in the clinical aspects of the disease they are studying. These opportunities enable identification of therapeutic targets with the greatest relevance to human disease, inform preclinical safety and efficacy testing, and define preclinical outcome measures that parallel clinical metrics. Furthermore, this program familiarizes trainees with the complexities of the regulatory processes required for human clinical trials and the business of taking research products to patients. A novel, and highly successful, aspect of the training is coaching in communication, conflict resolution, interpersonal and group interactions skills. These skills are important both for success in working in interdisciplinary teams and for helping in the success and retention of underrepresented minority students. Taken together, these opportunities will prepare students for careers inside and outside academia and will advance the NIH goals of enhancing biomedical research in the context of health and human services as a whole. The program we have implemented thus fills a significant training gap in the translational application of stem cell biology to neurological disorders, not typically met by traditional neurobiology, stem cell or clinical graduate programs.