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Fig. 6
L-leucine partially improved developmental deficiencies of ESCO2 depleted embryos in a TOR pathway-dependent manner.
(A). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-MO (4 ng) and immediately separated for D-Leu or L-Leu incubation (10 mM) for 2 d.p.f. L-Leu supplement partially rescued development of ESCO2-morphant embryos at a gross level. Scale bar = 200 μm. Animals were categorized as mildly affected, severely affected, or dead to further quantify the rescue. (B). The number of severely malformed and lethal embryos was quantified for ESCO2-depleted embryos in the presence of D-Leu or L-Leu supplement at 4 days post fertilization. A total of 73-115 embryos were quantified per condition (ESCO2-5mis with D-Leu (n = 73), ESCO2-MO with D-Leu (n = 114), ESCO2-5mis with L-Leu (n = 97), and ESCO2-MO with L-Leu (n = 115)). (C). Embryos (1–2 cells) were injected with ESCO2-MO (4 ng) and immediately transferred to L-Leu incubation at different concentrations. L-Leu supplement ameliorated the developmental defects of ESCO2 morphant embryos in a dosage-dependent manner. While the image is representative, about 20 embryos were analyzed per group. Bar = 200 µm. (D). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-MO (4 ng) and immediately separated into D-Leu or L-Leu incubation (3 mM) for 24 h.p.f.. By Western blot analysis, L-Leu supplement partially rescued phosphorylation of S6 in the ESCO2-MO embryos. S6 and tubulin serve as loading controls. Each sample contains ~100 embryos. (E). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-MO (4 ng), and immediately separated into D-Leu or L-Leu incubation (10 mM) for 3 d.p.f., in the presence or absence of 200 nM rapamycin. While the image is representative, about 15 embryos were analyzed per group. Rapamycin curtails L-Leu rescue of ESCO2-morphants, and enhances malformation of ESCO2-depleted embryos. Bar = 200 μm. (F). WT and ESCO2 mutant embryos (1–2 cells) were incubated with 10 mM D-Leu or L-Leu for 4 d.p.f., and photographed. L-Leu treatment partially rescued development of ESCO2 mutant embryos. While the image is representative, about 10 embryos were analyzed per group. Bar = 200 μm. (G). WT and ESCO2 mutant embryos (1–2 cells) were incubated with 10 mM D-Leu or L-Leu for 2 d.p.f. Western blotting shows L-Leu treatment partially restored phosphorylation of S6 in ESCO2 mutant embryos, but p53 elevation persists. S6 and tubulin serve as loading controls.