Mitotically driven cytoskeletal reorganization governs zebrafish left-right organizer detachment from EVL and lumen morphogenesis.

Left-right asymmetry in vertebrate embryos is established by the left-right organizer (LRO), with the zebrafish Kupffer's vesicle (KV) providing a tractable model for studying de novo epithelial morphogenesis. During KV formation, dorsal forerunner cells (DFCs) initially form polarized attachments to the enveloping layer (EVL) before reorganizing into multicellular rosettes that precede lumen formation. Here, we show that while DFC-EVL junctions form independently of mitosis, early cytokinetic events are required for remodeling these contacts. Live imaging and targeted laser ablation reveal that cytokinetic bridges and their associated microtubule bundles recruit actin, seed rosette centers, and promote the transition from external to internal epithelial organization. Disruption of early, but not later, DFC divisions impairs actin accumulation, rosette coalescence, KV detachment from the EVL, and lumenogenesis. These findings identify a temporally restricted role for cytokinesis in organizing cytoskeletal architecture and reveal how division history directs epithelial tissue assembly during LRO development.