Gradient Formation of the TGF-β Homolog Dpp

genes spalt (sal) and optomotorblind (omb) at different Summary cytonemes (Ramírez-Weber and Kornberg, 1999) shape the Dpp activity gradient. These studies point to the Secreted morphogens such as the Drosophila TGF-␤ homolog Decapentaplegic (Dpp) are thought to spread necessity of directly monitoring the distribution and trafficking of Dpp to approach the cellular and molecular through target tissues and form long-range concentration gradients providing positional information. Us-basis of gradient formation. Here we show Dpp tissue distribution, subcellular localization, and trafficking in ing a GFP-Dpp fusion, we monitored a TGF-␤ family member trafficking in situ throughout the target tissue the receiving cells. and forming a long-range concentration gradient. Evidence is presented that long-range Dpp movement Results involves Dpp receptor and Dynamin functions. We also show that the rates of endocytic trafficking and degra-Tagged Dpp Is a Functional Ligand dation determine Dpp signaling range. We propose a GFP was fused to the Dpp mature peptide (Panganiban model where the gradient is formed via intracellular et al., 1990) which is processed in secreting cells (Figure trafficking initiated by receptor-mediated endocytosis 1a). It resulted in a stable chimeric GFP-Dpp protein of the ligand in receiving cells with the gradient slope (see Experimental Procedures) allowing us to monitor controlled by endocytic sorting of Dpp toward recy-the traffic of secreted Dpp in the target tissue by GFP cling versus degradation. fluorescence after fixation or in vivo (Figure 1b). To determine whether GFP-Dpp signals like endogenous Dpp, Introduction we performed rescue experiments (Figures 1c–1g) in the dpp d12 /dpp d14 mutant (Masucci et al., 1990). This mutant During pattern formation and organogenesis, cells ac-lacks dpp and sal target gene expression in the wing quire information about their location within a field of (Lecuit et al., 1996) (Figure 1d), dies during early pupara-equivalent cells and differentiate according to their position , and fails to differentiate epidermal adult structures tion. In Wolpert's positional information model (Wolpert, (Bryant, 1988). GFP-Dpp driven by a Dpp wing promotor 1969), a " form generating substance " (Turing, 1952), a in dpp d12 /dpp d14 elicits Sal expression over the same morphogen, emanates from its source, diffuses across range as endogenous Dpp (about 15 cell diameters in the the target field and forms a long-range concentration posterior compartment; Figures 1c and 1e) and restores gradient. Receiving cells interpret the gradient by acti-normal epidermal structures (Figures 1f and 1g). These vating target gene expression at discrete …