The ceramide synthase 2b gene mediates genomic sensing and regulation of sphingosine levels during zebrafish embryogenesis

Abstract

Sphingosine-1-phosphate (S1P) is generated through phosphorylation of sphingosine by sphingosine kinases (Sphk1 and Sphk2). We show that sphk2 maternal-zygotic mutant zebrafish embryos (sphk2MZ) display early developmental phenotypes, including a delay in epiboly, depleted S1P levels, elevated levels of sphingosine, and resistance to sphingosine toxicity. The sphk2MZ embryos also have strikingly increased levels of maternal transcripts encoding ceramide synthase 2b (Cers2b), and loss of Cers2b in sphk2MZ embryos phenocopies sphingosine toxicity. An upstream region of the cers2b promoter supports enhanced expression of a reporter gene in sphk2MZ embryos compared to wildtype embryos. Furthermore, ectopic expression of Cers2b protein itself reduces activity of the promoter, and this repression is relieved by exogenous sphingosine. Therefore, the sphk2MZ genome recognizes the lack of sphingosine kinase activity and up-regulates cers2b as a salvage pathway for sphingosine turnover. Cers2b can also function as a sphingolipid-responsive factor to mediate at least part of a feedback regulatory mechanism.

Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish

“…Our total ceramide content for 7 dpf zebrafish was 1.0 ± 0.1 nmol/mg protein (Table 1), or 145 ± 7 pmol/15 larvae. Previous measurements in 6 hpf-7 dpf zebrafish ranged from ~30 pmol/15 embryos to ~22 nmol/15 larvae 16,41,66 . These variabilities may be partially explained by differences in the ceramides targeted, larval age and strain, but could also be a consequence of sample processing.…”

“…Sphingolipids are required for early vertebrate development, and disrupted sphingolipid metabolism can lead to defects in organogenesis 74,75 . Morpholinos against the sphingosine-1-phosphate receptors s1pr1 and s1pr2 severely inhibit intersegmental vessel angiogenesis 65 , and delayed epiboly is observed in a maternal zygotic mutant of ceramide synthase 2b 41 . Zebrafish carrying a nonsense mutation in 3-ketodihydrosphingosine reductase, an enzyme of the de novo ceramide biosynthetic pathway, exhibits hepatosplenomegaly that progresses to steatosis and liver injury 66 .…”

“…A lipid class that is significantly implicated in LSDs is sphingolipids 36 . Sphingolipids comprise a major lipid class with essential functions in embryogenesis, signalling, apoptosis, immunity and membrane integrity 37–41 . Given their involvement in all major aspects of cell biology, loss-of-function mutations in almost any sphingolipid catabolic enzyme can lead to pervasive substrate accumulation, multiple-organ pathologies and early mortality 36 .…”

Dietary rescue of lipotoxicity-induced mitochondrial damage in Peroxin19 mutants

“…Interestingly, a recent study could show that the zebrafish Schlank homologue ceramide synthase 2 reacts to increased levels of sphingosine by enriching in the nuclear membrane compartment, similar to Schlank in response to coconut oil supplementation [ 42 ]. The enzymes responsible for producing the sphingosine precursor sphinganine are the serine palmytoyltransferases Spt1 and Spt2 (the latter is known as Lace in Drosophila ).…”

Sphingosine kinases protect murine embryonic stem cells from sphingosine-induced cell cycle arrest

“…Sphingolipids were extracted after the addition of internal standards and quantified by liquid chromatography with tandem mass spectrometry as described previously. 28 2.10 | Quantitative fluorescent in situ hybridization and karyotyping Cells for quantitative fluorescent in situ hybridization (QFISH) of telomeres were cultured, induced, and sorted as described above. Two hours before dissociation for FACS sorting, cells were treated with 50 μL/mL Colcemid (Invitrogen), and submitted postsort to the Molecular Cytogenetics Core at the Memorial Sloan Kettering Cancer Center.…”