Mechanisms of Embryonic Patterning and Lineage Specification

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0318768EtkinsI. Intellectual merit of the proposed studyIn the frog, Xenopus laevis, localized maternal RNAs and proteins play important roles in early embryogenesis. In a large scale screen for vegetally localized maternal RNAs the cDNA encoding fatvg was cloned. Fatvg is localized through both the METRO (Early) and late pathways and is associated with the germ plasm in cleavage stage embryos. It is related to a group of mammalian proteins that include adipocyte differentiation related protein (ADRP) and TIP47 that have diverse functions including transport of lipid bodies, regulation of signaling pathways, and molecular trafficking within a variety of cell types. A loss of function analysis of fatvg using antisense oligodeoxynucleotides demonstrated that fatvg plays a dual role in dorsal/ventral patterning and in the specification of the germ cell lineage. The data also show that this dual effect is through the inability of maternal dorsalizing and germ cell determinants to segregate properly in fatvg?depleted embryos. This indicates that both the determinants specifying the germ cell lineage and the dorsal/ventral axis may utilize a common mechanism in segregating their products in the embryo or that their functions are regulated through a common pathway. It is likely, that the mechanism involved in the transport of dorsal and germ cell determinants relies on the movement of vesicles that carry these factors. This is consistent with the observations that fatvg protein is detected coating vesicle-like structures located at the vegetal cortex. This represents a new and novel mechanism involved in regulating germ cell specification and dorsal?ventral patterning. Based on the potential function of fatvg related proteins in molecular trafficking in mammalian cells it is hypothesized that fatvg is a key player in initiating the molecular pathways responsible for germ line specification and dorsal ventral patterning through its role regulating molecular trafficking. This hypothesis will be tested by carrying out the following specific aims: A. To analyze the co localization of fatvg protein with dorsalizing factors and germ cell determinants on cellular structures and how this is altered in fatvg depleted embryos; B. To determine the relationship between fatvg containing vesicles, the cytoskeleton and molecular motors; C. To identify the interacting proteins through which Fatvg functions.II. Broader Impact resulting from the proposed study.The role of fatvg in both patterning and cell lineage specification through its role in molecular trafficking is unique and will illistrates a novel mechanism that integrates both processes. This would be an important contribution to the understanding of early developmental mechanisms and would have a broad impact on the fields of developmental biology, cell biology, and embryology. In addition to the potential scientific advance, the proposed project will involve the training of undergraduates (summer students in the laboratory), graduate students, and postdoctoral scientists. This is an important aspect of our work as scientists and how workers in this field relate to society.