Functional dissection of the "Drosophila melanogaster" fibroblast growth factor signalling pathway in branching morphogenesis of the developing tracheal system

Fibroblast Growth Factor (FGF) signalling is involved in numerous developmental processes ranging from cell determination to mitogenesis, and cell survival to cell migration. Interestingly, the same signalling pathway is used reiteratively throughout development and the question regarding the intracellular specificity is raised. Little is known about the intracellular signalling events of the FGF signalling pathway leading to specific cellular responses. Since the FGF signal is essential throughout embryonic and adult development and plays a role in many pathogenical processes, it is important to identify the factors, which determine the differential responses. We were interested to investigate the specificity of FGF signalling in a developmental context in which the signal induces directed cell migration, a cellular phenomenon that relies on changes of the cytoskeletal architecture. During gastrulation in early embryonic development, but also during the formation of organs in mammals and in Drosophila, FGFs have been shown to act as chemoattractants and guide cells toward their targets. In these contexts, FGF signalling has been shown to induce filopodia, which are long cellular extensions containing parallel actin bundles. Using Drosophila tracheal and mesodermal cell migration as model systems, we found that the intracellular domain of the two Drosophila FGF receptors (FGFRs) Breathless (Btl) and Heartless (Htl) can be replaced by the equivalent domains of Torso and EGFR, and yet these hybrid receptors will rescue cell migration in btl or htl mutant embryos, respectively. These chimeric receptors rescued cell migration even in the absence of Downstream-of-FGFR (Dof), a scaffolding protein that has been shown to be essential for FGF signalling in Drosophila. Thus, Dof acts specifically in the FGF signalling pathway. The functional characterization of Dof has demonstrated that Dof is indeed a FGFR specific phosphotarget and forms a complex with both FGFRs, but it is not a substrate of Torso. We performed a functional deletion analysis of the Btl receptor to define the interaction domains of Dof and other putative adapter proteins essential in the process of cell migration. Deletion of all putative interaction domains outside of the kinase domain did not affect the rescue capacity of the truncated Btl receptors in vivo suggesting that the kinase domain is sufficient for transmitting the signal. In line with this interpretation, results from S2 cell culture experiments revealed that Dof interacts with the kinase domain, and it does so independently of the activation state of the receptor. Surprisingly, in S2 cells, Btl receptors lacking the C-terminus did not auto-phosphorylate, as consequence we could not observe phosphorylation of Dof. We assume, that the short C-terminus is required for conformational changes of the kinase activation loop upon dimerization of the receptors to enable trans-phosphorylation. Dof belongs to a distinct family of adapter proteins than its functional homologue, the vertebrate FGFR adapter protein FRS2 that has been shown to constitutively interact with the juxtamembrane domain of the FGFRs. We could show that the human FGFR2, when expressed in the tracheal system, is only able to rescue cell migration defects effectively in the presence of Dof. These results suggest that Dof is able to interact with human FGFRs. At present, there is no evidence for a FRS2 homologue in Drosophila that might act as substitute for Dof. Upon receptor activation, Dof recruits the phosphatase Corkscrew (Csw), the Drosophila Shp2 homologue. Csw recruitment represents an essential step in FGF induced cell migration and transcriptional activation via the Ras/MAPK cascade. However, our results indicate that the activation of Ras is not sufficient to activate the migration machinery in tracheal and mesodermal cells. Ectopic activation of the Ras/MAPK cascade partially rescued tracheal cell migration in btl or dof mutant embryos. But high levels of sustained activation of Ras or the MAPK in wild-type tracheal cells did not disturb the migratory behaviour of the cells in contrast to ectopic activation of Branchless (Bnl), the Drosophila FGF homologue, which completely impaired primary branch outgrowth. In a wild-type tracheal system, MAPK activity is restricted to the tracheal tip cells. Single cell rescue experiments indicate that Bnl induces the migratory response exclusively in the tip cells of the outgrowing tracheal branches; the stalk cells are pulled forward by cell-cell adhesion contacts. The small GTPases of the Rho family have been shown to regulate cytoskeletal rearrangements. For tracheal development, Dcdc42 could function in collaboration with Drac in the regulation of actin dynamics according to our experiments. Additional proteins linking either Dof or Csw to the small GTPases have to be identified.