Figure 2. Cilia-targeted GECIs reveal the presence of ICOs in the LRO of zebrafish that are leftward-biased and dependent on Pkd2 and ciliary motility.

A–F. Spatial mapping of ICOs in the zebrafish LRO. (A, C, E) Representative fluorescent live images of LRO from vehicle-injected (A), pkd2 morphant (MO) (C) and c21orf59 morphant (E) zebrafish embryos at the 1-somite stage expressing ratiometric GECI (arl13b-GCaMP6/mCherry; ratiometric signal shown and false-colored with rainbow intensometric scale). White arrowheads indicate LRO cilia at resting state, while magenta arrowhead (A) highlights an ICO inducing a cytosolic calcium wave. (B, D, F) Rose diagrams depicting the spatial distribution and mean percentage of cells per embryo displaying ICOs in the total LRO of vehicle (B), pkd2 MO (D) and c21orf59 MO (F) embryos spanning the entire course of LRO development (bud to 16-somite stage). Rings correlate with mean percentage of cells exhibiting calcium oscillations per embryo from bud to 16-somite stage (n = 10 embryos at all stages for pkd2 and vehicle; n = 5 embryos for c21orf59 and vehicle). Control and experimental samples were acquired in a pair-wise manner and analysis was performed on time-lapse recordings spanning bud to 16-somite stage. Control samples for c21orf59 morphants are not shown as the distribution and frequency was equivalent to controls for pkd2 morphants. Left-sided calcium, blue. Right-sided calcium, orange. G. Intraciliary calcium at the LRO exhibits oscillation-like dynamic. Ratiometric intensity over time plot of a single cilium exhibiting ICOs in vehicle (blue), pkd2 MO (magenta) and c21orf59 MO (green) LROs at the 1–4 somite stage. Each plot was aligned post-acquisition to the first detected ICOs (indicated by black arrowhead) and thresholded (indicated by black horizontal line) to facilitate analysis of calcium oscillation dynamics. A: Anterior, P: Posterior, L: Left, R: Right. Scalebars in A,C,E: 10 μm. See also Figures S2 and S3; and Movies 1–3.