Florence L. Marlow, Ph.D. - US grants

DESCRIPTION (provided by applicant): Asymmetry in oocytes is a well-documented and conserved feature among animals including vertebrates and humans. In vertebrates, the earliest indicator of cell polarity is an asymmetric aggregate, known as the Balbiani body, that includes organelles, proteins, and, in some animals, mRNAs encoding germline determinants. In non-mammalian vertebrates, this early asymmetry is known to indicate the animal-vegetal axis, but the relationship between the Balbiani body and the animal-vegetal axis in mammals is not understood. Although the animal- vegetal axis is the first axis to form in vertebrates, and is crucial for normal development of the embryonic axes that form later in development, its specification is poorly understood. In a maternal-effect genetic screen we isolated 2 alleles of bucky ball (buc), mutants that lacks oocyte asymmetry and fails to establish the axes in embryos. The Buc protein does not contain any characterized or known functional domains based on sequence comparison, but other vertebrates including humans have bucky ball genes. The buc gene, and our mutant alleles provide the first genetic access and a unique entry point to the developmental pathway regulating oocyte polarity. Here three aims are proposed to study how cell polarity is established and maintained in the vertebrate ovary. 1) We will test the hypothesis that buc specifies the oocyte axis upstream or at the level of Balbiani body assembly. 2) We have identified Buc interacting proteins. We will study these interacting proteins, and conduct rescue based structure function analysis to identify Buc functional domains to understand the mechanism by which Buc regulates animal-vegetal polarity. 3) We will determine which factors mediate asymmetric buc mRNA localization and contribute to oocyte polarity. Understanding how the Balbiani body, a conserved oocyte asymmetric structure, forms in vertebrates will break new ground in the field of axis formation. Studies of the genetic and molecular control of axis formation in zebrafish will clarify the mechanisms establishing these earliest oocyte asymmetries, which are conserved. In humans, mutations disrupting genes required to specify oocyte polarity or the first embryonic axis are expected to result in failed implantation or miscarriage due to severe developmental abnormalities. These most severe birth defects often are not detected in humans. In model systems such as zebrafish where fertilization and development of the embryo occur externally every egg that is produced can be examined for developmental abnormalities. Thus, this vertebrate genetic system allows access to maternally regulated developmental processes. An improved understanding of the essential maternal genes regulating early embryonic development in zebrafish will provide insight into the basis of birth defects and miscarriage, and facilitate comparison with human proteins. Studies of the Buc pathway are expected to be particularly relevant to abnormalities arising in very early pregnancy since buc mutant females produce eggs that are fertilized, but fail to specify the embryonic germ layers or axes. Completing these studies will provide insight into how Bucky ball regulates Balbiani body formation and oocyte asymmetry. These studies represent a first step toward deciphering the genes and mechanisms, mediating an evolutionarily conserved feature of primary oocyte development that is predicted to play fundamental roles in fertility, and in some vertebrates, establishment of the embryonic axes. PUBLIC HEALTH RELEVANCE: Prior to zygotic genome activation, vertebrate development depends on maternally supplied factors. However, the identity of the essential components and the molecular mechanisms underlying many maternally driven processes are not known. Mutations disrupting strict maternal-effect genes are viable. The mutant females are overtly normal, due to maternal function supplied by their mother. However, all of their progeny display the mutant phenotype regardless of their genotype. Although maternal products are essential for vertebrate development, only a small fraction of the vast numbers of vertebrate genes with maternal expression have been experimentally evaluated through genetic or by interference technologies. In each of these cases, insufficient maternal contribution results in early embryonic arrest, or profound developmental abnormalities. Similar genetic defects in humans would be expected to result in failed implantation or miscarriage before pregnancy is detected. Ten to twenty percent of known pregnancies result in miscarriage;however, when combined with undetected pregnancies, the actual percentage of pregnancies ending in miscarriage is estimated to be as high as 40-50% of all pregnancies, according to The March of Dimes, The American College of Obstetricians and Gynecologists, The Mayo clinic, and the National Institutes on Child Health and Development. Our research goal is to elucidate the genetic pathways and cell biological events that establish the first embryonic axis. We will use a combination of genetic, molecular, and cell biological approaches in the zebrafish model system. In humans, loss of function mutations in genes whose products are required to specify the first embryonic axis are expected to result in miscarriage due to severe developmental abnormalities. Studies of the genetic and molecular control of axis formation in zebrafish will clarify the genetic basis of animal-vegetal axis formation, potentially illuminating the genetic basis of human birth defects, and early miscarriages of unknown etiology.

?A Transgenic System for Targeted Ablation of Reproductive and Maternal-Effect genes in Zebrafish? Maternally provided gene products regulate the earliest events of embryonic life, including formation of the oocyte that will develop into an egg, and eventually an embryo. Forward genetic screens have provided invaluable insights into the molecular regulation of embryonic development, including essential contributions of some genes whose products must be provided to the transcriptionally silent early embryo for normal embryogenesis, maternal-effect genes. However, other maternal-effect genes are not accessible due to their essential zygotic functions during embryonic development. To identify these factors it is necessary to bypass the early requirement of the gene so potential later functions of the gene can be investigated. Methods to circumvent these zygotic lethal phenotypes in the zebrafish were pioneered by Brian Ciruna, Alex Schier, and Erez Raz. Their germline replacement approach takes advantage of the early separation of somatic and germline lineages in zebrafish to generate animals with a normal somatic composition and a mutant germline through host germline ablation and transplantation (replacement) with mutant donor germ cells. This strategy allows the animal to survive to produce mutant gametes, which can be examined for reproductive and maternal-effect phenotypes. Although this approach has been applied to examine the function of specific genes, thus far, no systematic germline replacement screen of the existing large collection of zebrafish zygotic lethal mutations has been reported. We are proposing a reverse genetic system designed to identify genes whose reproductive and maternal-effect functions are masked by their essential zygotic roles in early embryogenesis. Identifying these regulators is essential to fill the large gaps in our understanding of the mechanisms and molecular pathways contributing to fertility and maternally regulated developmental processes. The goal of the studies proposed here is to develop and apply a robust and efficient genetic method to mutate the host germline and to conduct a proof of concept screen of genes known to have adult reproductive and maternal-effect phenotypes, including vasa, Kif5Ba, and bucky ball.  

SUMMARY The oocyte, when fertilized has the capacity to generate every cell in an organism. Efforts to understand and harness the reprogramming potential of this ?mother of all stem cells? gave rise to today's stem cell research field. Despite their unique developmental potential, oocytes are highly specialized and differentiated cells. A key step in oocyte development is the transition from a mitotic germline stem cell that in most animals is specified prior to sexual differentiation to a meiotic germ cell that develops as an oocyte in females or sperm in males. Development of the oocyte is a prolonged developmental time-period characterized by highly conserved periods of meiotic activity, arrest, and substantial oocyte growth and maturation to yield a fertilizable egg. Before the first meiotic division the oocyte stops producing new RNAs. Consequently, control of programs vital to egg production and fertility rely heavily on regulation by proteins, called RNA binding proteins, that bind to RNAs and regulate their activity. Reproductive success relies on proper establishment and maintenance of sexual identity. Defects in germ cell differentiation can lead to infertility or germ cell tumors. Identifying RNA binding protein targets, and determining how they act in oocyte development is essential to fill the large gaps in our understanding of the mechanisms and molecular pathways that preserve female sex identity of the germ cells and fertility. Our long- term goal is to determine the mechanisms that regulate sex-specific programing of germ cells. We utilize the zebrafish, a powerful vertebrate genetic system to examine the mechanisms and identify genes that are crucial for successful fertility, maintenance of germ cell identity and ovarian reserve, and thus may define new molecular pathways that, when defective, can result in reduced fertility, premature ovarian insufficiency, polycystic ovary syndrome, or cancers in humans. In this proposal, mechanisms regulating sex-specific differentiation of germ cells and fertility will be tested using a combination of genetic loss of function and over expression approaches.