Follicular patterning Directed by Janus Kinase Signaling

Morphogens are substances with the ability to directly induce, over a distance, different cell fates based on levels of the molecule received. The mechanisms by which morphogens move from the source to target cells is particularly interesting. The currently funded research has sought to understand the distribution and activity of the putative morphogens Unpaired and Unpaired3, ligands of the Janus kinase signaling pathway in Drosophila melanogaster. Previous work in the lab identified an essential role for JAK signaling in oogenesis: anterior-posterior patterning of the follicle cells that surround the developing egg. This patterning is controlled by a gradient of JAK activation across the follicular epithelium that is highest at the termini and lower toward the center. The level of JAK activation in the follicle cells specifies which of five potential epithelial fates will be adopted. The Upd and Upd3 ligands are secreted by the polar cells, which are located at the anterior and posterior termini, and activate JAK signaling in the follicular epithelium. The specific aims of the currently funded research are to: I) determine the mechanism by which graded JAK activation is established in the follicular epithelium and 2) determine the mechanisms by which the Upd and Upd-like molecules are distributed and reach their targets in the ovary. Research during the current granting period has focused on the roles of the Upd and Upd3 ligands in JAK signaling, particularly in oogenesis. Of relevance to the present supplementary funding request, we have generated the first mutations in the Upd3 to examine its function. Loss ofUpd3 activity causes the reduction or loss of the eye and an abnormal outstretched posture to the wings that prevents flight. This phenotype is shared with a unique set of mutations, called outstretched (os), which we have found to be distinct from both upd and upd3. Assessment of this relationship would be pursued in a renewed grant. Surprisingly, loss of upd3 does not have a dramatic influence on follicle cell differentiation. However, Upd and Upd3 share similar roles for in eye and wing development. Furthermore, preliminary analysis of expression of these genes in imaginal discs shows overlapping expression in the developing wings and eyes. These data suggest that Upd and Upd3 act in concert, at least in some tissues. Current work in the lab seeks to extend the studies described above. In particular, the coexpression of Upd family ligands in all tissues examined suggests that there is coordination of their activities. Preliminary results using bimolecular fluorescence complementation (BiFC) support the hypothesis that the Upd ligands physically interact to carry out their functions. In BiFC, two proteins suspected of interacting are fused to two different halves of a fluorescent protein. If the proteins interact, fluorescence may be detected due to the close proximity of the complementary halves of the fluorescent protein. The rationale for choosing this method to assay protein interaction is that it can be performed as a competition between alternate binding partners, permitting excellent quantification of relative binding affinities. Furthermore, it can provide spatial information regarding subcellular and tissue sites of interaction that cannot be ascertained from biochemical approaches. Because there are no reports using this method in Drosophila, we have generated BiFC vectors that can be used for either in vitro or in vivo studies. Constructs for Upd and Upd3 have been made to test for homo- and heterodimer (or multimer) formation. Preliminary experiments in Drosophila Schneider (S2) cells strongly suggest that both proteins complex with themselves and with each other. Co-transfection of one plasmid with Upd or Upd3 fused to the YFP N-terminus, one plasmid with Upd or Upd3 fused to the CFP C-terminus, and a plasmid expressing GAL4 from the su(Hw) promoter all result in strong fluorescence complementation detectable within the cell prior to secretion (see Fig. 1). As a negative control, we tested BiFC of an Upd fusion with a Dpp (BMP ligand) fusion. Both proteins are secreted, so should be within the same intracellular compartments. Fluorscence is nearly undetectable in the control (Fig. 1), suggesting that presence of the complementary fluorescent protein peptides in the same subcelular location is insufficient to permit complementation.