Fig. S1

Developing spinal secondary motor neurons (SMN) generate spontaneous intracellular Ca²⁺ waves. (A) A SMN (arrow) loaded with calcium green-1 dextran in the spinal cord of a zebrafish embryo at 20–21 hpf. Dorsal is to the left and caudal is at the bottom. Images were captured at 0.2 Hz for 30 min, and fluorescence intensity was normalized to baseline. Fluorescence is displayed on a pseudocolor scale, where purple represents the lowest intensity and red represents the highest intensity. Changes in fluorescence intensity in a SMN are plotted as a function of time. Ca²⁺ waves were identified as fluorescence transients greater than 20% of ΔF/F0 (dashed lines), more than two times the SD of the baseline, and >20 s in duration, calculated as the width at half-maximum. (B) Duration and frequency of Ca²⁺ waves in SMN. n = 15–20 neurons from seven to nine embryos for each time period; values are means ± SEM. *P < 0.05.