Abstract
In mammals, the primitive ectoderm is an epithelium of polarized cells that differentiates into all embryonic tissues. Our study shows that in primitive ectoderm cells, the sphingolipid ceramide was elevated and co-distributed with the small GTPase Cdc42 and cortical F-actin at the apicolateral cell membrane. Pharmacological or RNA interference-mediated inhibition of ceramide biosynthesis enhanced apoptosis and impaired primitive ectoderm formation in embryoid bodies differentiated from mouse embryonic stem cells. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog. Ceramide depletion prevented plasma membrane translocation of PKCzeta/lambda, its interaction with Cdc42, and phosphorylation of GSK-3beta, a substrate of PKCzeta/lambda. Recombinant PKCzeta formed a complex with the polarity protein Par6 and Cdc42 when bound to ceramide containing lipid vesicles. Our data suggest a novel mechanism by which a ceramide-induced, apicolateral polarity complex with PKCzeta/lambda regulates primitive ectoderm cell polarity and morphogenesis.
Highlights
In mammals, the primitive ectoderm is an epithelium of polarized cells that differentiates into all embryonic tissues
Replenishment of ceramide or substitution with S18 resulted in partial restoration of the proportion of embryoid bodies (EBs) with well organized primitive ectoderm and defined cavity (Fig. 1, D and E, and supplemental Fig. S1, D and E). These results showed that inhibition of ceramide biosynthesis impaired primitive ectoderm morphogenesis, which was compensated by exogenously added ceramide or a ceramide analog
The fluorescence signal was strictly co-distributed with that of ceramide, suggesting that ceramide was associated with pPKC/ (Fig. 5A). This assumption was supported by the detection of a ceramideto-pPKC/ FRET signal Taken together, these results suggest that ceramide is associated with PKC/ at the apicolateral membrane and that ceramidebound PKC/ is phosphorylated at threonine 403/410
Summary
The primitive ectoderm is an epithelium of polarized cells that differentiates into all embryonic tissues. Our studies have shown that the membrane sphingolipid ceramide regulates apoptosis during embryonic stem (ES) cell differentiation [1,2,3,4,5]. This regulation is initiated by direct physical interaction of ceramide with atypical PKC or [5]. The formation of the primitive ectoderm is followed by the removal of cells that are not in contact with the primitive endoderm derived basal lamina These cells die by apoptosis giving rise to the proamniotic cavity, a process termed cavitation [7]. Their inhibition has been linked to neural tube defects in humans and animals caused by the disruption of sphingolipid biosynthesis, indicating the significance of ceramide or its derivatives for developmental processes prior to or during neurulation [14, 15]
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