Insights into the zebrafish left-right organizer's centrosomes and cilia via volume electron microscopy.

The zebrafish left-right organizer (LRO), Kupffer's vesicle (KV), is a ciliated epithelial organ for which its three-dimensional architecture underlies symmetry breaking during embryonic development. While KV cilia have been extensively studied by light microscopy, their ultrastructural organization, heterogeneity, and spatial patterning within the intact organ remain incompletely defined. Here, we establish volumetric electron microscopy (vEM) as a platform for high-resolution, three-dimensional analysis of KV architecture. Using vEM, we reconstructed nearly an entire KV at nanometer resolution, enabling comprehensive assessment of cilia, centrioles, appendages, rootlet fibers, and associated vesicles within their native tissue context. Ultrastructural analysis revealed heterogeneity in centrosome architecture associated with KV cilia, including variability in centriole composition and the presence of distal and subdistal appendages as well as rootlet fibers. In addition, cilia were frequently associated with distinct classes of membrane-bound vesicles, including ciliary-associated vesicles and ciliary-associated dense vesicles. Beyond describing KV ultrastructure, this dataset illustrates how vEM can be leveraged, while also highlighting important caveats related to sampling depth, developmental staging, and interpretation of centriole loss versus remodeling. Collectively, this work provides a foundational vEM resource for the zebrafish LRO and establishes a framework for integrating volumetric ultrastructural analysis with developmental and functional studies of ciliated tissues.