Lysosomes are Required for Early Dorsal Signaling in the Xenopus Embryo

Abstract

Lysosomes are the digestive center of the cell and play important roles in human disease, including cancer. Previous work has suggested that late endosomes, also known as multivesicular bodies (MVBs), and lysosomes are essential for canonical Wnt pathway signaling. Sequestration of Glycogen Synthase 3 (GSK3) and of {beta}{square}catenin destruction complex components in MVBs is required for sustained canonical Wnt signaling. Little is known about the role of lysosomes during early development. In the Xenopus egg, a Wnt-like cytoplasmic determinant signal initiates formation of the body axis following a cortical rotation triggered by sperm entry. Here we report that cathepsin D was activated in lysosomes specifically on the dorsal marginal zone of the embryo at 64-cell stage, long before zygotic transcription starts. Expansion of the multivesicular body (MVB) compartment with low-dose Hydroxychloroquine (HCQ) greatly potentiated the dorsalizing effects of the Wnt agonist Lithium chloride (LiCl) in embryos, and this effect required macropinocytosis. Formation of the dorsal axis required lysosomes, as indicated by brief treatments with the vacuolar ATPase (V-ATPase) Bafilomycin A1 inhibitor at the 32-cell stage. Inhibiting the MVB-forming machinery with a dominant-negative point mutation in Vacuolar Protein Sorting 4 (Vps4-EQ) also interfered with the endogenous dorsal axis. The Wnt-like activity of the dorsal cytoplasmic determinant Huluwa (Hwa), and that of microinjected xWnt8 mRNA, also required lysosome acidification and the MVB-forming machinery. We conclude that lysosome function is essential for early dorsal axis development in Xenopus. The results highlight the intertwining between membrane trafficking, lysosomes, and vertebrate axis formation. SignificanceThe dorsal axis of the vertebrate Xenopus embryo is established by an early Wnt signal generated by a rotation of the cortex of the egg towards the opposite side of the sperm entry point. In this study, we report that lysosomal Cathepsin D becomes activated on the dorsal marginal zone of the embryo already at the 64-cell stage, and that this asymmetry is enhanced by increasing Wnt signaling levels. We present experiments showing that lysosome activity, macropinocytosis, and multivesicular body formation are required for the dorsal signal provided maternally in the egg, and for twinning by microinjected huluwa and Wnt8 mRNA. The results indicate that the cell biology of lysosomes plays a fundamental role in vertebrate development.