Fig. 7

L-leucine rescues many aspects of development in ESCO2-depleted embryos.

(A). 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). After 5 d.p.f., the embryos were stained with Alcian blue to detect cartilage development. Scale bar = 200 μm. While the image is representative, about 15 embryos were analyzed per group. (B). WT or ESCO2 mutant embryos were treated with D-Leu or L-Leu (10 mM). After 5 d.p.f., the embryos were stained with Alcian blue to detect cartilage development. While the image is representative, about 10 embryos were analyzed per group. L-leucine partially rescued head size and cartilage formation in (A) and (B). (C). Cranial development was quantified for the data in (A) using the sum of the pq (palatoquadrate) cartilage and mc (Meckel′s cartilage) divided by cranial length, as indicated with lines in (B). The measurement was done on 3 embryos per group. P<0.01, ESCO2-MO with D-Leu treatment vs ESCO2-5mis with D-Leu treatment; P<0.05, ESCO2-MO with L-Leu treatment vs ESCO2-MO with D-Leu treatment. (D). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-MO (2 ng), and immediately separated for D-Leu or L-Leu incubation (10 mM). After 2 d.p.f., the embryos were stained with acridine orange to detect apoptotic cells. Scale bar = 200 μm. (E). The number of apoptotic cells was quantified. P<0.0001, ESCO2-MO with D-Leu treatment vs ESCO2-5mis with D-Leu treatment; P = 0.0006, ESCO2-MO with L-Leu treatment vs ESCO2-MO with D-Leu treatment. (F). Embryos (1–2 cells) were treated as in (A). After 2 d.p.f., single cell suspensions of 10 embryos were generated in triplicate by mashing and filtering in PBS/10% NCS through cell strainers (100 μm, BD Falcon). Cells were resuspended in PBS/10% NCS and suspensions used in Caspase-Glo 3/7 Assays (Promega) according to the manufacturer′s instructions. Luminescence was measured after 75 minutes on a multi-detection microplate reader. L-Leu treatment suppressed caspase 3/7 activation in the ESCO2-MO embryos. P<0.0001, ESCO2-5mis with D-Leu vs ESCO2-MO with D-Leu; P<0.0001, ESCO2-MO with L-Leu vs ESCO2-MO with D-Leu. (G). Embryos (1–2 cells) were treated as in (A). Embryo length was measured every day up to 4 d.p.f. with a mini-ruler under a Leica Stereoscope for ESCO2-MO and 5-mismatched controls. L-Leu treated ESCO2 morphants had a significantly longer body length compared with ESCO2 morphants treated with D-Leu (P<0.0001, 2-way ANOVA). The measurements were performed from head to tail for 10 embryos per group.