The body plan emerges from information laid down in three axes during development: the anterior-posterior, dorsal-ventral, and proximal-distal (PD) axes. The PD axis specifies values at increasing distances from the central body, and all the limbs require such information. The fruit fly, Drosophila melanogaster, has proven to be a valuable model system to explore axis formation, and work in the investigator's laboratory has focused on the PD axis specifically. Mutations in the Drosophila four-jointed (fj) gene result in loss of discrete positional values in the middle of the PD axis of the legs and wings. The fj gene encodes a novel, conserved transmembrane protein that can be cleaved in vivo to form a secreted polypeptide, a potential signaling ligand. Four genes have been shown to interact genetically with fj: the dachs (d) approximated (app), abelson (abl), and enabled (ena) genes. This proposal is concerned with the further analysis of fj and its interacting partners, with the long-term objective of understanding both how Fj functions and how this relates to the genetic control of limb morphogenesis and the specification of a PD axis. Four issues will be addressed. (1) The relative importance of the membrane and secreted forms of Fj will be explored using site-directed mutagenesis to create altered forms of the protein in transgenic flies. (2) Alternative hypotheses of how Fj functions will be tested by the controlled loss or ectopic expression of Fj in small patches of tissue in the imaginal discs. (3) Domain-specific antibodies will be used to study the subcellular distribution of the FJ protein, its potential association with cellular junctions, and its relationship to interacting genes. (4) d mutants have phenotypes almost indistinguishable from those of fj, and genetic studies suggest it is the best candidate for a Fj receptor. Consequently, d will be cloned making use of chromosomal mapping and gamma deficiencies already in hand.
