Wg secreted by conventional Golgi transport diffuses and forms Wg gradient whereas Wg tethered to extracellular vesicles do not diffuse

s Wingless (Wg)/Wnt family proteins are essential for animal development and adult homeostasis. Drosophila Wg secreted from the dorsal-ventral (DV) midline in wing discs forms a concentration gradient that is shaped by diffusion rate and stability of Wg. To understand how the gradient of extracellular Wg is generated, we compared the secretion route of NRT-Wg, an artificial membrane-tethered form of Wg that is supposedly not secreted but still supports fly development, to that of wild-type Wg. We found that wild-type Wg is secreted by both conventional Golgi transport and via extracellular vesicles (EVs), and NRT-Wg can be also secreted via EVs. Furthermore, wild-type Wg secreted by Golgi transport diffused and formed Wg gradient but Wg-containing EVs did not diffuse at all. In case of Wg stability, Sol narae (Sona), a metalloprotease that cleaves Wg, contributes to generate a steep Wg gradient. Interestingly, Wg was also produced in the presumptive wing blade region, which indicates that NRT-Wg on EVs expressed in the blade allows the blade cells to proliferate and differentiate without Wg diffused from the DV midline. We propose that EV-associated Wg induces Wg signaling in autocrine and juxtaposed manners whereas Wg secreted by Golgi transport forms gradient and acts in the long-range signaling, and different organs differentially utilize these two types of Wg signaling for their own development.