Fig. 2

Effect of GCP on GALC activity in zebrafish. A) Extracts of 96 hpf zebrafish embryos (50 μg/sample) and of adult zebrafish brain (20 μg/sample) were incubated at room temperature with increasing concentrations of GCP in the presence of the GALC substrate LRh-6-GalcCer. After overnight incubation, the GALC product LRh-6-Cer was separated from its substrate by TLC, visualized under an ultraviolet lamp, and photographed. B) Extracts of zebrafish embryos, zebrafish, and murine adult brains, and recombinant human GALC expressed in HEK293 cells (rGALC) were incubated with GCP as in A. The GALC product LRh-6-Cer was quantified following its extraction from the silica gel plates. Data are the mean ± S.E.M of 3–4 independent experiments. C) In two independent experiments, zebrafish embryos at 1–2 cell stage were injected with 160 pmoles of GCP/embryo or vehicle. At 96 hpf, 8 GCP-treated and 8 vehicle-treaded animals were pooled and processed for GALC activity assay as in panel B. Inset: Image of the TLC analysis performed on the pools of vehicle (-) or GCP (+) treated animals. P, paired Student’s t test. D) Light sheet 3D reconstruction of Tg(neurod1:EGFP)ia50 embryos injected at 6 hpf with 160 pmoles/embryo of GCP or vehicle and visualized at 24 hpf. After image acquisition, 3D reconstructions were performed using the Arivis software (Zeiss) and exported as a single snap with the same compression settings. White arrows indicate the midbrain and eye regions of the embryos.