The goal of the proposed research is to understand how communication between cells contributes to developmental processes. Genetic, molecular, and cell biological techniques will be employed to study the cell-cell interactions in the fruit fly Drosophila melanogaster that coordinate the migrations of the follicle cells during their production of the eggshell (or chorion). We will characterize a network of genes required to produce the two dorso-lateral respiratory appendages, or filaments (the filament pathway genes rhino, zeppelin, Ras1, homeless, and bullwinkle). Genetic analyses will define the mutational spectrum of each gene, and molecular analyses will identify transcripts and provide potential functional information from sequence data. Analysis of mosaics generated by mitotic recombination and pole cell transplantation, in situ hybridization to ovarian tissue sections, and Northern blot analysis will provide spatial and temporal information concerning their function. Examination of fixed and stained egg chambers that carry a dorsal follicle cell-specific marker will help distinguish between models in which our filament path genes affect dorsal/ventral positional cues, or alter the cell migration process itself. Observation of the follicle cell migrations in in vitro culture will determine if physical defects are present in these lines. To define the interactions among the filament path genes, we will continue our genetic and molecular epistasis tests. To refine our definition of their exact position in the filament pathway, we will create an activated Ras1 construct that can be used to order loss of function mutations in other genes. We will also employ the activated Ras1 line to test models that address the mechanism by which the follicle cells interpret the spatial coordinate system and thereby position the dorsal filaments correctly. To flesh out our knowledge of the filament path process, we will identify new genes in the filament pathway by recovering dominant enhancers and suppressors of specific Ras1 alleles. We will also identify interacting genes by examining changes in enhancer trap expression patterns following introduction of various filament path mutations. We expect to identify a cohort of genes that are unique in that they couple positional cues derived from cell-cell interactions with the coordinated migration of populations of cells. Our filament pathway analyses, however, will be generally applicable to understanding many other cell signaling-cell migration processes. Our proposed experiments combine genetic, molecular and cell biological techniques to ascertain the steps by which an initiating signal is translated into directed cell movement and coupled to the ordered expression of the chorion genes to yield the dorsal filaments. These studies therefore examine the process by which cells transmit, receive, and interpret positional information and direct the ordered process of development.
