Tube formation, the folding of flat epithelial sheets into curved structures, is a basic process in the development of organs such as the lungs, kidneys, heart, neural tube, and gut. The most common permanently disabling birth defects in the U.S. result from errors in the development of tubes. To understand the mechanisms that control tube formation, this project investigates a tube-forming process that occurs in developing egg chambers of the fruit fly, Drosophila melanogaster. In the fly ovary, certain follicle cells within the epithelium that surrounds the oocyte reorganize from a curved sheet into a pair of elongated tubes. This process is remarkably similar to mammalian neural tube formation, which creates the spinal cord. Genetic studies show that signals originating from small populations of cells, both outside and within the tubes, regulate tube closure and elongation. Faulty signaling produces open or stunted tubes, similar to human defects that cause spina bifida or fetal death. Analyses of live and fixed tissue show that, within the epithelium, subsets of cells respond differently to the signals, inducing unique behaviors that shape the tubes. To identify and characterize these signaling molecules and the factors within cells that facilitate their distinctie responses, this proposal uses an innovative adaptation of a magnetic-bead approach to purify the sub-populations of cells and then evaluate the protein and RNA content in each cell type. Preliminary data implicate cytoskeletal regulators, a novel class of growth factors, and cell-type-specific transcription factor complexes in regulating tube-cell migration. By comparing molecules between different cell types, and between wild type and mutants, this work will identify signaling components and reveal why cells respond differently to the signals. Functional analyses in vivo using powerful genetic and live-imaging tools will elucidate the mechanisms that contribute to errors in tube formation. Since tube formation is highly conserved between invertebrates and vertebrates, these studies will give insight into human development and disease.