Multiple Wnt signaling pathways direct epithelial tubule interconnection in the regenerating zebrafish kidney

Epithelial tubule fusion is fundamental for kidney morphogenesis. Differentiating nephron tubules interconnect with collecting system epithelia to generate a lumenal pathway for fluid excretion. In the adult zebrafish kidney, nephrogenesis occurs as a regenerative response to injury and provides a model to explore cell signaling pathways required for tubule interconnection. We show that canonical Wnt signaling at the junction between two tubules induces a mesenchymal, invasive cell phenotype and is required, along with Src kinase and rac1, to generate basal cell protrusions. The Wnt ligands wnt9b and wnt4 are both required for new nephron formation after injury. Mutation in wnt4 or treatment with the canonical Wnt inhibitor IWR1 blocks formation of basal protrusions in forming nephrons. Mutation in the Wnt receptor frizzled9b reveals a fusion-associated non-canonical Wnt pathway that acts to 1) restrict canonical Wnt gene expression, 2) drive Rho kinase-dependent apical constriction of epithelial cells, and 3) position basal protrusions and generate orthogonal tubule lumenal connections. As a result, frizzled9b mutant nephrons fail to fully interconnect with target distal tubules. Our results indicate that canonical and non-canonical Wnt signaling interact in the same cells to orient and drive tubule interconnection in the regenerating zebrafish kidney.