Fig. 4

SepN and RyR3 are required for calcium signaling at the KV. (A and B) sepN and ryr3 transcripts are present in and around the KV (indicated by arrow) at the base of the notochord (ntc). (C–F) Visualization of free calcium in the region of the KV in 5- to 8-somite embryos. Images shown represent a single confocal section, taken from a dorsal view at the plane of the maximal apparent diameter of the KV. (C) Without calcium-sensitive dye, fluorescence is not detected. (D) Elevated levels of free calcium are detected on the left compared with the right side of the KV in WT embryos. (E) Loss of ryr3 function results in a dramatic overall reduction of free calcium. (F) Loss of sepN function results in complete loss of asymmetric distribution of free calcium. (G) For quantitative analyses, left- and right-side intensity values were determined for each embryo by integrating pixel values within symmetric boxed areas abutting the KV midline as indicated in C-F. Left/right intensity ratios were determined for each embryo [WT controls, n = 17; rhodamine dextran-injected (RD), n = 11; sepN MO-injected, n = 22; ryr3 MO-injected, n = 19] and average ratios are graphed. *, significant difference from WT ratio (P < 0.001 using paired Student′s t test). Error bars are ± SD.