Ononiwu, F.; Mikolaj, M.; Dell, C.; Shamil, A. W.; Narayan, K.; Hehnly, H.

In the zebrafish left-right organizer (LRO), the Kupffers Vesicle (KV), cilia extend from all cells into the fluid-filled lumen, but their structural diversity and contribution to morphogenesis remain incompletely defined. We hypothesized that cilia are required for KV development and may exist in distinct structural subtypes. Using a newly engineered transgenic line (sox17:Cry2-GFP), we optogenetically disrupted the intraflagellar transport protein IFT88 in KV progenitors via blue light-induced clustering of CIB1-RFP-IFT88. This perturbation impaired ciliogenesis and disrupted lumen formation, supporting a critical role for cilia in KV morphogenesis. To assess ciliary architecture, we used volume electron microscopy (vEM) to generate a high-resolution 3D ultrastructural map of mature KVs. Only 70.1% of cilia retained both mother and daughter centrioles, suggesting centriole elimination may occur in this tissue. Among centrioles present, 33.9% had distal appendages, 91.8% had subdistal appendages, and only 5.08% exhibited rootlet fibers. Cilia were also associated with membrane-bound vesicles, including spatially biased ciliary-associated vesicles (CaVs) and dense vesicles (CaDVs). These findings demonstrate that KV cilia are structurally diverse and spatially patterned, revealing a previously unappreciated level of complexity in LRO organization and providing new insight into how ciliary specialization may contribute to left-right axis specification.