Fig. 4.

nSMase2 regulates ceramide production in the plasma membrane of neural crest cells. A. Schematic illustrating displays presented in panels B (high magnification section through premigratory neural crest in the dorsal neural tube) and C (equatorial and ventral surface planes of the same field of view of explanted neural crest cells). B. Three focal planes from a single z-stack show that nSMase2-FLAG localizes to the plasma membrane together with GPI-eGFP in the Sox9-positive premigratory neural crest cells in vivo (closed arrows). C-D. nSMase2-FLAG also localizes to the plasma membrane (closed arrow) and filopodia (open arrow) in explanted neural crest cells ex vivo (C), and in U2OS human osteosarcoma cells (D). E. Embryos were electroporated with the indicated reagents and neural crest cells were explanted and cultured ex vivo. After the start of migration, explants were stained for ceramide and single-cell ceramide levels were quantitated. F. Dot plot of ceramide intensity values, with each dot representing a single cell (n = 193 Control MO, n = 134 nSMase2 MO, n = 173 H2B-RFP, n = 235 nSMase2-FLAG cells) and overlayed bars displaying the mean and 95% CIs. ****P < 0.0001; Mann–Whitney U test. (All scale bars represent 10 µm.)