Oocytes maintain precise cytoplasmic organization, making them an excellent system to study mechanisms of cell polarity. Oocyte polarity establishes the developmental axes of fly, frog and fish embryos; thus, understanding oocyte polarity is critical to understanding embryonic development. The first marker of oocyte polarity in zebrafish is the Balbiani body (Bb): a transient aggregate of mRNA, ribonucleoprotein particles, mitochondria and other membrane bound organelles. The Bb carries the germ line determinants and polarized mRNAs from the presumptive vegetal side of the nucleus to the vegetal cortex and defines the vegetal half of the developing oocyte. This is a novel mechanism generating oocyte polarity, but despite the Bb's conservation across many vertebrates, including mice and humans, little is known about it. The first goal of this proposal is to perform functional analysisof the subcellular localization and protein structure of bucky ball (buc), the only gene known to be vital for Bb formation. Female zebrafish homozygous mutant for buc produce oocytes with no Bb; the resulting eggs are nonviable and are symmetric exhibiting animal character radially, demonstrating a complete loss of animal-vegetal polarity. buc mRNA undergoes specific localization during oocyte development: first, in stage I oocytes buc localizes to the Bb and then later, in stage III oocytes, buc localizes to the animal pole. It is not clear how the localizationof the mRNA affects Buc function, but buc transgenes with a truncated 3' UTR rescue poorly the mutant, suggesting that post- transcriptional regulation of Buc is important to its function. Buc homologs are present in many vertebrate groups, including amphibians, birds and mammals. However, Buc contains no predicted domain structures. I hypothesize that buc mRNA localization via the 3' UTR is an early critical step in its function, accompanied by action of one or more distinct functional protein domains of Buc to form the Bb. The second goal of this proposal is to place Buc within a greater structural and functional context, which is vital for building a comprehensive mechanism for Buc function in establishing animal-vegetal polarity. Given the lack of predictable functional domains within Buc and the complexity of the Bb, Buc is expected to act with other proteins to form the Bb and polarize the oocyte. However, the lack of a complete set of Bb proteins makes it difficult to construct a mechanism for Buc's function. Using a proteomics approach I have identified a large number of proteins within the Bb. I will screen a set of these novel Bb proteins for function within the Bb and for binding with Buc to find new functional elements of the Bb and new binding partners of Buc. In this proposal, I will characterize the structure/function of Buc and functionally characterize new protein elements of the Bb to lay the groundwork for understanding the mechanism of Bb formation and function.
Oocyte polarity determines the embryonic axes in lower vertebrates, and depends on a transient structure called the Balbiani body, which translocates to the vegetal oocyte cortex, where it disassembles and deposits a dorsal axis determinant and germ cell determinants. Because basic questions about how the Balbiani body forms and regulates its cargo remain unanswered, this project aims to complete the first steps that will build a framework for understanding this novel mechanism generating cell polarity. By understanding the Balbiani body, we may uncover new molecular mechanisms by which cells break symmetry and generate distinct subcytoplasmic regions, a process key to preventing metastasis in cancer.
|Elkouby, Yaniv M; Jamieson-Lucy, Allison; Mullins, Mary C (2016) Oocyte Polarization Is Coupled to the Chromosomal Bouquet, a Conserved Polarized Nuclear Configuration in Meiosis. PLoS Biol 14:e1002335|