Analysis of Kupffer's vesicle in zebrafish embryos using a cave automated virtual environment

Previous studies show that cilia in Kupffer's vesicle (KV) generate a counterclockwise flow of fluid and provide convincing evidence that this flow regulates left/right asymmetry. We hypothesized that the distribution of cilia in KV leads to this directional flow. However, there are limitations in determining the localization of structures when viewing a three-dimensional (3-D) image on a 2-D computer screen. We analyzed the distribution of KV cilia in the Cave, an immersive virtual environment that displays stacks of confocal images in 3-D. We found 80% of the cilia are located on the dorsal surface and 20% were located on the ventral surface of the vesicle. We confirmed the ventral location of some cilia by electron microscopy. There is an asymmetrical distribution of cilia on the dorsal surface, with the anterior one third containing 50% and the posterior one third containing 20% of the cilia. This dorsal-anterior patch could explain the directionality of the flow, and could drive local differences in flow rate.