Rabconnectin-3a Regulates Vesicle Endocytosis and Canonical Wnt Signaling in Zebrafish Neural Crest Migration

Abstract

A novel role for Rabconnectin-3a in endosome maturation regulates Wnt signaling, migration, and fate specification in zebrafish neural crest cells.

Abstract

Cell migration requires dynamic regulation of cell–cell signaling and cell adhesion. Both of these processes involve endocytosis, lysosomal degradation, and recycling of ligand–receptor complexes and cell adhesion molecules from the plasma membrane. Neural crest (NC) cells in vertebrates are highly migratory cells, which undergo an epithelial–mesenchymal transition (EMT) to leave the neural epithelium and migrate throughout the body to give rise to many different derivatives. Here we show that the v-ATPase interacting protein, Rabconnectin-3a (Rbc3a), controls intracellular trafficking events and Wnt signaling during NC migration. In zebrafish embryos deficient in Rbc3a, or its associated v-ATPase subunit Atp6v0a1, many NC cells fail to migrate and misregulate expression of cadherins. Surprisingly, endosomes in Rbc3a- and Atp6v0a1-deficient NC cells remain immature but still acidify. Rbc3a loss-of-function initially downregulates several canonical Wnt targets involved in EMT, but later Frizzled-7 accumulates at NC cell membranes, and nuclear B-catenin levels increase. Presumably due to this later Wnt signaling increase, Rbc3a-deficient NC cells that fail to migrate become pigment progenitors. We propose that Rbc3a and Atp6v0a1 promote endosomal maturation to coordinate Wnt signaling and intracellular trafficking of Wnt receptors and cadherins required for NC migration and cell fate determination. Our results suggest that different v-ATPases and associated proteins may play cell-type-specific functions in intracellular trafficking in many contexts.

Author Summary

The neural crest is a highly migratory population of embryonic cells, which requires Wnt signaling at several stages to promote migration and cell fate decisions. Intracellular trafficking of Wnt receptors and associated proteins can affect the timing and intensity of Wnt signaling. An obvious question is whether proton pumps and/or their partner proteins that are associated with intracellular vesicles might have a role in intracellular trafficking, Wnt signaling, and cell migration/adhesion. In this study we demonstrate such a role for Rabconnectin-3a, a protein associated with the vacuolar-ATPase (v-ATPase) proton pump complex. Loss of Rabconnectin-3a in zebrafish embryos disrupts the maturation of endocytic vesicles in neural crest cells, which has two effects: (1) decreasing Wnt signaling in these cells before migration and (2) increasing Wnt signaling after migration. Prior to migration, endosomes that fail to mature reduce Wnt signaling in neural crest cells and disrupt the localization and expression of cadherins, membrane-bound cell adhesion molecules required for these cells to initiate an epithelial-mesenchymal transition. At later stages, however, Wnt receptors accumulate at the membranes of unmigrated neural crest cells due to defective endocytosis, which correlates with high levels of Wnt signaling. Interestingly, Rabconnectin-3a-deficient neural crest cells that fail to migrate become pigment cells, presumably due to elevated Wnt signaling. Rabconnectin-3a may have a conserved role in endosomal maturation, Wnt signaling, and cell migration in many other cell populations.