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Dev Dyn
2008 Jan 01;2371:112-23. doi: 10.1002/dvdy.21406.
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Cloning and functional characterization of two key enzymes of glycosphingolipid biosynthesis in the amphibian Xenopus laevis.
Luque ME
,
Crespo PM
,
Mónaco ME
,
Aybar MJ
,
Daniotti JL
,
Sánchez SS
.
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Gangliosides are a subfamily of complex glycosphingolipids (GSLs) with important roles in many biological processes. In this study, we report the cDNA cloning, functional characterization, and the spatial and temporal expression of Xlcgt and Xlgd3 synthase during Xenopus laevis development. Xlcgt was expressed both maternally and zigotically persisting at least until stage 35. Maternal Xlgd3 synthase mRNA could not be detected and showed a steady-state expression from gastrula to late tailbud stage. Xlcgt is mainly present in involuted paraxial mesoderm, neural folds, and their derivatives. Xlgd3 synthase transcripts were detected in the dorsal blastoporal lip, in the presumptive neuroectoderm, and later in the head region, branchial arches, otic and optic primordia. We determined the effect of glycosphingolipid depletion with 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP) in mesodermal layer. PPMP-injected embryos showed altered expression domains in the mesodermal markers. Our results suggest that GSL are involved in convergent-extension movements during early development in Xenopus.
Figure 3. Biochemical and cellular characterization of XLCGT and XLGD3 synthases. A: CHO-K1 cells transiently expressing Xlcgt were immunostained with antibody to HA (red) and examined by the intrinsic fluorescence of YFP from Sial-T2-YFP, a Golgi marker (pseudocolored green) or Iip33-YFP, a endoplasmic reticulum (ER) marker (pseudocolored green). Merge, overlap of both XLCGT and marker signals (orange-yellow). B: XLCGT catalyze the formation of glucosylceramide. TLC analysis was carried out with cell lysates. Lane 1: Mock transfected wild-type CHO-K1 cells. The enzymatic reaction was stopped at t = 0; Lanes 2 and 3: Mock transfected wild-type CHO-K1 cells (60 min of incubation). Lanes 4 and 5: Transiently transfected CHO-K1 cells expressing XLCGT (60 min of incubation). Cer, ceramide; GlcCer, glucosylceramide; SM, sphingomyelin. C: CHO-K1 cells transiently expressing XLGD3 synthase were immunostained with antibody to HA (red) and examined by the intrinsic fluorescence of YFP from Sial-T2-YFP, a Golgi maker (pseudocolored green) or Iip33-YFP, an endoplasmic reticulum (ER) marker (pseudocolored green). Merge, overlap of both XLGD3 synthase and marker signals (orange-yellow). Single confocal sections were taken every 0.7 mu m parallel to the coverslip. Images are representative of the majority of the observed transfected cells. Scale bar = 10 mu m.
Figure 5. Expression of Xlcgt during Xenopus development. Nieuwkoop-Faber stages of embryogenesis are indicated. A,B: Xlcgt is expressed in the dorsal structures of stages 15-18 embryos. C: At stages 25-28, Xlcgt is expressed in the notochord. Inset: Transversal section (as indicated in C) shows strong expression in the notochord. D: At later stages (stage 35), the pattern of expression was maintained in the notochord and a faint expression domain was detected in the cephalic region. n, Notochord; hm, headmesoderm.
Figure 6. Expression of the Xlgd3 synthase gene during Xenopus development. Nieuwkoop-Faber stages of embryogenesis are indicated. A: Xlgd3 synthase is faintly expressed in the dorsal mesoderm of stage-11 embryos. B: Histological section showing expression in the dorsal blastopore lip (as indicated in A). C: Stage-13 embryos show expression in dorsal structures (neural plate, notochord, and paraxial mesoderm). D: Transverse section of C. E: Stage-16 embryo. F: Transverse section of E. G: Stage-19 embryo showing expression of GD3s in the neural tube, notochord, and somites. H: Transverse section of G. I,J: In stage-22 embryos, GD3s is strongly expressed in the neural tube and notochord. K: Stage-35 embryos, the Xlgd3 synthase mRNA was detected in the head region, branchial arches, otic and optic primordia. This transcript was also localized in the notochord and was faintly expressed in the somites. bl, blastopore lip; nt, neural tube; n, notochord; s, somites; opp, optic primordium; otp, otic primordium; ba, branchial arches.
Figure 8. Glycosphingolipids depletion affects the localization of mesodermal markers. Expression of Xbra in stage-11 control embryos (A) and PPMP-treated embryos (A prime ). Expression of chd in stage-12 control embryos (B) and PPMP-treated embryos (B prime ). Expression of gsc in stage-12 control embyos (C) and PPMP-treated embryos (C prime ).
Glycosphingolipids depletion affects the localization of mesodermal markers. Expression of t in stage-11 control embryos and PPMP-treated embryos.
