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.

Wingless (Wg)/Wnt 家族蛋白对于动物发育和成人体内平衡至关重要。果蝇从翼盘背腹（DV）中线分泌的 Wg 形成浓度梯度，该浓度梯度由 Wg 的扩散速率和稳定性决定。为了了解细胞外 Wg 的梯度是如何产生的，我们将 NRT-Wg 的分泌途径与野生型 Wg 的分泌途径进行了比较，NRT-Wg 是一种人工膜束缚形式的 Wg，据说不分泌但仍支持果蝇发育。我们发现野生型 Wg 既可以通过常规高尔基体运输也可以通过细胞外囊泡 (EV) 分泌，NRT-Wg 也可以通过 EV 分泌。此外，由高尔基运输分泌的野生型 Wg 扩散并形成 Wg 梯度，但含有 Wg 的 EVs 根本没有扩散。在 Wg 稳定的情况下，Sol narae (Sona)，一种切割 Wg 的金属蛋白酶，有助于产生陡峭的 Wg 梯度。有趣的是，Wg 也在假定的机翼叶片区域产生，这表明在叶片中表达的 EV 上的 NRT-Wg 允许叶片细胞在没有 Wg 从 DV 中线扩散的情况下增殖和分化。我们提出 EV 相关 Wg 以自分泌和并列方式诱导 Wg 信号传导，而高尔基体转运分泌的 Wg 形成梯度并在远程信号传导中起作用，并且不同的器官差异地利用这两种类型的 Wg 信号传导来进行自身发育。