Fig. 4

The sphk2^MZ embryos express strikingly high levels of cers2b transcripts.

(A) Analysis of ceramide synthase and ceramidase genes by qRT-PCR shows that cers2b is significantly upregulated in sphk2^MZ embryos (p=0.019) compared to wildtype embryos at 2 hpf. (B) Representative wildtype or (C) sphk2^MZ embryos following injection with 5.3 ng of a cers2b translation blocking morpholino (MO), alongside (D) embryos derived from a sphk2^MZ; cers2b^+/- incross. For wildtype embryos, 155/161 (96%) completed normal gastrulation, epiboly, and appeared normal at 2 dpf. Following injection into sphk2^MZ embryos, 60/288 or 21% from 4 separate experiments recapitulated the early gastrulation lethality phenotype (p=0.0154). Correspondingly, 43/190 or 23% of embryos derived from crossing sphk2^MZ; cers2b^+/- adult fish recapitulated early gastrulation lethality phenotype by 9 hpf. (E) Sphk2 morphant embryos fail to upregulate cers2b transcripts and (F) did not vary in ceramide levels when compared to those injected with control morpholino. (G) Embryos exposed to 5 uM sphingosine in culture similarly fail to upregulate cers2b transcript levels and (H) showed no differences in ceramide accumulation. (I) There were no significant differences in sphingosine (Sph) or S1P (Sph-1P) levels in embryos with maternally deposited Sphk2, consistent with normal development. Embryos were harvested for both qPCR and lipid measurements at 6 hpf. All experiments were repeated at least three times with equivalent results.