Wnt signaling is implicated in a variety of developmental and pathological processes. The molecular mechanisms governing the secretion of Wnt ligands remain to be elucidated. Wntless, an evolutionarily conserved multipass transmembrane protein, is a dedicated secretion factor of Wnt proteins that participates in Drosophila melanogaster embryogenesis. In this study, we show that Xenopus laevis Wntless (XWntless) regulates the secretion of a specific Wnt ligand, XWnt4, and that this regulation is specifically required for eye development in Xenopus. Moreover, the Retromer complex is required for XWntless recycling to regulate the XWnt4-mediated eye development. Inhibition of Retromer function by Vps35 morpholino (MO) resulted in various Wnt deficiency phenotypes, affecting mesoderm induction, gastrulation cell movements, neural induction, neural tube closure, and eye development. Overexpression of XWntless led to the rescue of Vps35 MO-mediated eye defects but not other deficiencies. These results collectively suggest that XWntless and the Retromer complex are required for the efficient secretion of XWnt4, facilitating its role in Xenopus eye development.The Wnt family of glycoproteins comprises one of the largest families of paracrine factors essential for embryonic development and adult tissue homeostasis (reviewed at the Wnt Homepage, http://www.stanford.edu/ϳrnusse/wntwindow .html). It regulates several aspects of biological processes, including cell fate specification, proliferation, migration, and polarity formation (29, 48). The Wnt signaling pathway is initiated by Wnt ligands secreted from Wnt-producing cells. The ligands bind to frizzled receptors and coreceptors expressed on the receiving cells. Wnt ligand perception (5,24,46,49), signaling cascades into receiving cells (8,27), and the consequences of gene expression (17) or cytoskeletal changes (36) are well documented in various contexts. However, relatively limited information is available about the processes in Wnt-producing cells and extracellular spaces. The establishment of the concentration gradient of the Wnt ligand in the extracellular space is mediated by lipoprotein particle formation (33). The Retromer complex is additionally required in Wnt-producing cells and for long-range secretion of Wnt (12). Porcupine is essential for posttranslational modifications, which may be essential for the proper folding and secretion of Wnt ligands (28).Recent achievements in Drosophila melanogaster genetics and genomic RNA interference screening have revealed the existence of a new component of the Wnt secretory pathway, specifically, a dedicated secretion factor of Wg designated Wntless (2), Evi (3), or Sprinter (16). Wntless is an evolutionarily conserved multipass transmembrane protein required solely for Wg secretion. Wntless is not essential for the palmitoylation of Wg, indicating that it does not act on functional Wg production, like Porcupine, another evolutionarily conserved multipass transmembrane protein. Wntless is a regulator of intracellular W...