Project Summary Vertebrate eggs lie dormant until fertilized, when a process called egg activation ensues that triggers the completion of meiosis and other processes that initiate embryonic development. These processes occur within the first several minutes after fertilization and depend on maternal factors supplied by the mother to the egg during oogenesis. Maternal factors exclusively drive early embryonic development in all animals, since the zygotic genome is not activated until at least one and typically several cell division cycles after fertilization. The large maternal contribution to vertebrate embryonic development is evident by the large size of eggs and early embryos compared to somatic cells (~10 to 1000 times greater, depending on the species). Moreover, unique features of this stage of development, such as acentrosomal meiotic divisions and mitotic spindles that cannot scale to the large cell sizes, necessitate unique maternal functions, not found in somatic cells. However, the roles that maternal factors play and how their vast supply is regulated spatiotemporally in the large egg and early embryo is little studied, despite its importance in reproductive success and fertility. The zebrafish provides an excellent model for studying the maternal regulation of development in vertebrates. Many maternally-regulated gene functions are conserved between zebrafish and mammals. Zebrafish eggs are externally fertilized, so are easily accessible, and their large size and translucency is advantageous for live imaging with fluorescent markers, which will be profited from here. A key event in egg activation is the completion of meiosis. At the end of oogenesis in vertebrates, the mature oocyte and egg are arrested in metaphase of meiosis II. The second meiotic division ensues when the egg is activated, thus generating the second polar body and a haploid complement of chromosomes to contribute to the zygote. Meiotic divisions are unique in oocytes and eggs as they lack the centrosome, the primary microtubule organizing center (MTOC) of a cell. While the meiotic acentrosomal MTOC (aMTOC) proteome has recently been described, little is known about how acentrosomal spindles are regulated and what normally restricts their nucleation in time and space. Here a zebrafish maternal- effect mutant named volcán will be studied, which displays multiple ectopic spindle-like MTs in the single cell blastodisc that cause a novel defect, the generation of numerous vesicle-like structures at the animal pole potentially polar body-like structures, but devoid of DNA. The dynamics, etiology in development, and nature of the spindle-like MT and vesicle-like structures abrogated by Volcan will be investigated, as well as the molecular nature of the volcan gene. The results of these Aims are expected to reveal the molecular identity of a key protein acting to restrict formation or organization of aMTOC spindle MTs in the egg to ensure a single polar body-like structur...