ESTABLISHMENT OF THE GERM CELL LINEAGE IN DROSOPHILA

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The long term objective of this application is to gain a molecular understanding of how the germ cell lineage of Drosophila is established during embryogenesis. Proper establishment and maintenance of cell fate is essential for normal growth and development of an organism. Defects in this fundamental process leads to cancers and birth defects. This proposal focuses on the genetic, molecular and biochemical examination of one gene known to be required for the establishment of the germ cell lineage, the germ cell-less gene. The germ cell-less gene encodes an mRNA that is localized to the germ plasm which contains the germ cell determinants. The function of germ cell-less, based on antisense, over-expression and ectopic localization studies is required for, and is capable of initiating some of the events of germ cell precursors, or ~pole cell~ formation. Analysis of the subcellular distribution of germ cell-less protein indicates that it is localized to the nuclear pores of the pole cell nuclei prior to, during and after germ cell fate is established. The germ cell- less gene therefore plays a central role in the establishment of the germ cell lineage. The initial focus of this project will be to verify the specificty of germ cell-less function for he establishment of the germ cell lineage by isolating strong and null mutants. Germ cell-less mutants will be obtained by a n lecular based, P-element reversion screen and a standard ems mutageneis based gentic screen. Germ cell- less mutants will be molecularly, genetically and phenotypically characerized to precisely define its role in germ cell formation and other developmental pathways. To explore its biochemical function, the germ cell-less protein will be mutated to make sub-cellular localization mutants to test if nucelar pore association is required for function. It will also be ectopically expressed in Drosophila tissue culture celis to assay for a role in regulating nuclear trafficking. To identify other genes which are required for germ cell specification, a yeast two-hybrid screen will be performed to identify proteins which physically interact with the germ cell-less protein. Genes identified by this screen will be evaluated by several criteria to determine if they have a role in the establishment of the germ cell lineage. In addition, a genetic interaction screen will be performed also to identify additional genes in this pathway.