Fig. 2

Excess sphingosine results in gastrulation defects and embryonic lethality, which is abrogated in the context of a sphk2^MZ mutation.

(A) Embryos cultured in DMSO display normal coverage of the yolk by the developing blastoderm during epiboly at 7 hpf. (B) Treatment of wildtype embryos with 5 μM D-erythro-sphingosine (the active naturally occurring isomer) or (C) 5 μM L-erythro-sphingosine (the inactive isomer) caused stalling of embryonic development during epiboly, rupture of the yolk membrane, and failure of the embryos to complete gastrulation. This phenotype was recapitulated by embryos treated with (D) 5 μM D-galactosyl-ß1–1'-D-erythro-sphingosine (Psychosine), (E) 10 μM dimethylsphingosine (DMS), and (F) 20 μM L-threo-Dihydrosphingosine (Safingol). Treatment of wildtype embryos with two chemical inhibitors of sphingosine kinases, (G) SKI (10 μM) or (H) BT-190 (3 μM) recapitulated the catastrophic embryonic phenotype. (I) The majority (144/173, 83%) of wildtype (WT) embryos treated with 5 μM sphingosine exhibited a lethal phenotype during gastrulation, whereas the majority (175/176, 99%) of the sphk2^MZ embryos treated with 5 μM sphingosine completed gastrulaton and early development, only subsequently displaying the cardiac bifid phenotype observed in sphk2^MZ embryos, with no additional defects. Control embryos were cultured in DMSO vehicle (1%). Shown are representative embryos, n is indicated, from at least three independent experiments.