Rab8a-positive vesicles transport Wnt8a along cytonemes in zebrafish embryogenesis.

Wnt signalling is a conserved pathway that orchestrates key developmental processes by regulating cell fate, proliferation, and tissue organisation. While the production and secretion of Wnt ligands are well characterised, less is known about how lipid-modified Wnts are delivered for long-range communication. Recently, cytonemes - actin-based signalling filopodia - have been identified as transporters of Wnts over distances to target specific cells in embryogenesis. Here, we characterise Rab8a-dependent vesicular trafficking as a crucial step in this process. Using human cell lines and zebrafish embryos, we show that Wnt ligands, such as Wnt8a, are transported with their carrier protein Wntless (Wls) in Rab8a-positive vesicles along cytonemes, where they fuse at the tips to enable hand-over and signal activation in neighbouring cells. Disruption of Rab8a function results in the intracellular retention of Wnt8a, reduces Wnt spreading and paracrine signalling, and alters embryonic patterning, consistent with reduced Wnt/{beta}-catenin function. Conversely, Rab8a activation enhances Wnt8a dissemination, leading to increased long-range signalling and, consequently, patterning defects in embryogenesis. Our findings uncover a dedicated intracellular trafficking route for Wnt delivery to and along cytonemes, offering new insights into how the spatial precision of Wnt spreading in vertebrate tissues is achieved.