Wnt signalling has important roles during development and in many diseases. As morphogens, hydrophobic Wnt proteins exert their function over a distance to induce patterning and cell differentiation decisions. Recent studies have identified several factors that are required for the secretion of Wnt proteins; however, how Wnts travel in the extracellular space remains a largely unresolved question. Here we show that Wnts are secreted on exosomes both during Drosophila development and in human cells. We demonstrate that exosomes carry Wnts on their surface to induce Wnt signalling activity in target cells. Together with the cargo receptor Evi/WIs, Wnts are transported through endosomal compartments onto exosomes, a process that requires the R-SNARE Ykt6. Our study demonstrates an evolutionarily conserved functional role of extracellular vesicular transport of Wnt proteins.

Wnt signaling pathways are important for multiple biological processes during development and disease. Wnt proteins are secreted factors that activate target-gene expression in both a short- and long-range manner. Currently, little is known about how Wnts are released from cells and which factors facilitate their secretion. Here, we identify a conserved multipass transmembrane protein, Evenness interrupted (Evi/Wls), through an RNAi survey for transmembrane proteins involved in Drosophila Wingless (Wg) signaling. During development, evi mutants have patterning defects that phenocopy wg loss-of-function alleles and fail to express Wg target genes. evi's function is evolutionarily conserved as depletion of its human homolog disrupts Wnt signaling in human cells. Epistasis experiments and clonal analysis place evi in the Wg-producing cell. Our results show that Wg is retained by evi mutant cells and suggest that evi is the founding member of a gene family specifically required for Wg/Wnt secretion.