Developmental Genetic Analysis of Fertilization Pathways in Drosophila

University Of Washington, Seattle WA

Investigators

Abstract

0211733 Wakimoto Fertilization begins with the initial contact of the sperm and the egg and ends with the joining of the paternal and maternal chromosomes in the embryo. The normal development of all sexually reproducing organisms requires the exquisite coordination of the activities of the sperm and egg during fertilization. Current knowledge of the mechanisms underlying fertilization has been obtained largely from morphological and biochemical studies of marine invertebrates and mammals. These studies have led to the discovery of a handful of molecules with confirmed roles in sperm-egg interactions in these organisms. The aim of the proposed project is to exploit advantages of a genetic system to identify functionally important fertilization molecules and pathways. A collection of male sterile lines of Drosophila melanogaster will be used to identify molecules expressed in sperm and essential for successful fertilization. Mutations that disrupt the sperm-egg binding or sperm activation after entry into the egg will be the focus of more extensive phenotypic and genetic mapping studies. These studies will define the types of genes that disrupt fertilization and their specificity during development.The proposed project will also involve the continued characterization two members of the "earliest developmental arrest" class of paternal effect genes. Mutations sneaky and space needle produce identical paternal effect defects, with mutant sperm failing to undergo the changes associated with activation after entry into the egg. The function of the Sneaky protein, a member of a newly discovered class of plasma membrane proteins, will be studied by localizing the wild type, tagged and mutated versions of the protein fusions. The effects of other paternal effect mutations, including space needle, on Sneaky function and localization will be analyzed to identify interacting molecules. Genetic and molecular mapping studies of the space needle gene will be performed to identify the gene and begin studies the function of its protein product. These studies work toward developing Drosophila into a powerful experimental system to investigate sperm-egg interactions. The results should be informative for assessing the degree to which the molecular pathways of fertilization are conserved among animals and used to mediate other cell-cell interactions during development. The overall aim of the proposed research is to understand the molecular mechanisms of sperm-egg interactions during fertilization. Dr. Wakimoto's strategy takes advantage of a collection of mutant strains of Drosophila in which males produce motile sperm that fail in the fertilization response. These mutants will be used to identify the molecular pathways that lead to successful sperm-egg binding and sperm activation. 1) To assess the number and types of mutants that are defective in sperm-egg interactions, they will: a) complete the phenotypic classification of putative fertilization defective and paternal effect mutants, b) determine the number of genes represented by the mutations through genetic complementation analysis and recombination mapping,. 2) To determine the molecular function of two genes, sneaky (snky) and space needle (spnl), which are required for sperm activation after entry into the egg, they will: a) define the subcellular localization and the functional domains of the Sneaky protein, the best characterized member of this class, b) assay the effects of another snky-like mutation, space needle (spnl) on the localization, modification and function of the Sneaky protein, c) complete the phenotypic characterization and mapping of spnl mutations toward identifying its protein product.