DESCRIPTION (provided by applicant): With the societal trend toward delaying pregnancy until advanced maternal ages, the reliance on assisted reproductive techniques is growing rapidly. A fundamental question for the field of In Vitro Fertilization (IVF) has been that of distinguishing healthy eggs and subsequent embryos from unhealthy ones. The inability to differentiate healthy embryos from unhealthy ones has resulted in the propensity for Fertility Clinics to transfer multiple embryos in the hopes that one will implant and result in a successful pregnancy. This practice has resulted in increased prevalence of multiple births that are inherently riskier and often result in premature delivery. In order to improve our understanding of
embryos suitable for implantation, it is imperative that we understand the molecular mechanisms that make a healthy egg develop into a healthy embryo. Formation and maturation of a developmentally competent egg requires that RNAs and proteins within the egg cytoplasm be poised to direct early zygotic development after fertilization. This is especially crucial when considering that the first cell divisions and cell fate specifications occur before the zygotic genome is active; thus, maternally deposited gene products are the sole drivers of early embryonic development. We use the zebrafish to study this process because zebrafish eggs are fertilized externally, and the embryos develop outside of the body and are optically transparent. This allows for clear visualization of early phenotypes that often result in embryonic
lethality. Additionally, zebrafish have excellent genetic tools and are exceptionally fertile, layig hundreds of eggs in a week, and are thereby a very powerful model for the genetic basis of egg and embryonic defects. In zebrafish and frogs, maternally deposited gene products include asymmetrically distributed RNAs and proteins responsible for establishing the germ line and the first embryonic axis. In mammals, it has not yet been determined if asymmetries in the egg are required for patterning of the embryonic axes. However, the oocytes of all animals (including humans) contain an asymmetrical structure, the Balbiani body (Bb), which is a transient subcellular aggregation of organelles and gene products. In zebrafish, the formation of the Bb is essential to deliver patterning molecules to one side of the egg. To date, only one gene has been demonstrated as necessary for Bb formation, the zebrafish bucky ball gene. We hypothesize that Bucky ball mediates assembly of the Balbiani body by interacting with RNA- binding proteins that, in turn, recruit patterning RNAs to this structure. To address this hypothesis, we will use classical genetics gain-of-function and loss-of-function approaches to assess the role of a conserved RNA- binding protein (RNAbp) in oocyte and embryonic patterning. Further, we will use biochemistry techniques to determine the nature of the interaction between Bucky ball and this RNAbp, and follow up this analysis with in vivo characterization on proteins lacking the interaction domains. Through imaging of transgenic and mutant zebrafish oocytes and embryos, this project will advance our understanding of oocyte patterning in vertebrates.