Analysis of Drosophila Fibroblast Growth Factor Functional Domains

The exciting Fibroblast Growth Factor (FGF) field lies at the crossroads of cell signaling, development, evolution, trafficking, physiology and human disease. A current challenge is to understand the mechanisms used by this signaling pathway to accomplish its myriad tasks in patterning the embryo, forming organs, and maintaining systems in the adult animal. My thesis work has focused on tackling this challenge in the model system of Drosophila melanogastor, the vinegar fly. By examining functional domains of Thisbe and Pyramus, FGF ligands in the fly, we have begun to understand the properties of Drosophila FGFs and the way in which they may contribute to regulation of FGF signaling. FGF ligands in vertebrates are small molecules that bind to a corresponding receptor through two immunoglobulin domains. The FGF ligands in Drosophila are predicted to be much larger molecules than their vertebrate homologs. Whether Drosophila FGFs bind to the receptor as full-length proteins or are first cleaved to smaller molecules was previously unknown. My thesis work addressed this question through experiments in Drosophila embryos and Drosophila cell culture. I found evidence that the N-terminal FGF-domain alone is capable of signaling by itself in the embryo. In addition, experiments in cell culture showed that Thisbe and Pyramus are secreted as small forms, presumably as a result of intracellular proteolytic cleavage. Cleaved forms for Thisbe and Pyramus were detected in embryonic extracts as well. The Ths ligand is also present outside the cell as a full-length form and this form may act to regulate the diffusion or activity of the ligand. Addition of the Thisbe C-terminus to the Pyramus N-terminus to make a Pyramus-Thisbe chimeric protein creates a protein that has reduced activity compared to Thisbe alone. The opposite Thisbe-Pyramus chimera creates a protein that has increased activity compared to Ths alone. Over the course of animal evolution the FGF superfamily has diversified in many ways. Understanding the mechanism of FGF signaling in Drosophila and comparing this to other Drosophilids, insects, and more distantly related animals will reveal the likely makeup of the ancestral FGF signaling system.