Project Summary Kidney disease is the 9th leading cause of death in the U.S. Because few therapies exist to prevent or slow progression, over 700,000 patients have End Stage Renal Disease. These patients are treated with dialysis or renal transplant, the latter resulting in markedly superior survival. However, kidney donors are limited and there is an important unmet need for strategies that enhance renal repair or generate new nephrons for renal replacement. Pluripotent stem cell derived organoids display key features of differentiated kidney tubules and glomerular structures in vitro, and we have shown that they generate patterned nephrons in vivo displaying kidney functions such as filtration and glucose uptake by the proximal tubule. To develop this technology for renal replacement, stem cell derived tubules must be connected to host tubules for urinary output. Our recent work in the zebrafish demonstrated that FGF signaling acts as a chemotactic signal to recruit and polarize cells at sites of new nephron formation and canonical Wnt signaling is required for invasive cell rearrangement to connect tubule lumens. Additional signaling pathways including non-canonical wnt signaling are also likely to play a role in tubule interconnection. To fully explore the requirements for tubule interconnection we have established a synergistic, three-part discovery platform comprising 1) genetic analysis of in vivo new nephron addition in the regenerating zebrafish adult kidney, 2) in vitro 3D cell culture analysis of mammalian epithelial fusion, and 3) in vivo stem cell-derived kidney organoid engraftment to a host mouse collecting system. We will combine these approaches to analyze multiple steps of the tubule fusion process involving 1) recruitment of nephron progenitor cells to target epithelia, 2) removal of intervening ECM/basement membranes, 3) patterned collective cell invasion of target epithelia, and 4) establishment of a continuous patent new lumen to convey the nephron filtrate. These studies will provide important new insights about an essential but understudied cellular mechanism that will be required for in vivo engraftment of new kidney tissue-based renal regeneration therapies.