Fig. 2

Cygb2 is expressed in cilia and through NO production regulates cilia structure.

a Kupffer’s vesicle (KV) at 8–10 somites stained with anti-acetylated tubulin (white) to visualize cilia and anti-Cygb2 (red) on whole mount embryos. The KV is outlined with a white dashed line. Scale bar = 10 μm. b Whole mount fluorescent in situ hybridization labeling dand5 (magenta) and cygb2 (yellow) transcripts using RNAscope. Image orientation: A- anterior, P- posterior, L- left, R- right. Scale bar = 10 μm. a, b show representative images of three experiments. On the right, rendering of a zebrafish embryo at 10 somites denoting the location of the KV. Panel generated with BioRender.com. c Immunostaining of KV cilia in wt and cygb2^801a with anti-acetylated tubulin antibody. On the right, average cilia length. Means are ± SD (n = 9–10 embryos). Student’s t test, two-tailed, ****P < 0.0001. Scale bar = 10 μm. d Transmission electron microscopy of KV cilia comparing wt and cygb2^801a. Scale bar = 100 nm. On the right, quantification of the number of central microtubules represented as the percentage of total number of cilia analyzed (n = 12–13 embryos). Chi-square test, ****P < 0.0001. e Schematic of the NO Analyzer (NOA). Panel generated with BioRender.com. f Chemiluminescence detection of NO production by triiodide assay using pools of 50 embryo (10 somites) lysates as samples comparing wt to cygb2^801a(above) and uninjected cygb2^801a to cygb2 mRNA injected cygb2^801a (below). g Quantification of nitrite levels normalized to protein amount. Samples represent pools of embryos (n = 3–14, each n is the lysate of 50 embryos) uninjected or injected with cygb2 mRNA (50 or 100 pg). Black bars represent wt embryos and blue bars represent cygb2^801a embryos. Means are ± SD. Student’s t test, two-tailed, *P < 0.05. Source data are provided as source data file.