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Fig. S7 The rescue of RBS zebrafish depends on ESCO2 mRNA, L-leucine and L-glutamine. (A, B). The specificity of the phenotype in ESCO2-morphant and mutant embryos was tested by injection of in vitro transcribed RNA encoding ESCO2 protein. Control injections were performed with in vitro transcribed control RNA. Scale bar = 200 μm. (C). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-Splice MO with control-mRNA or ESCO2-mRNA co-injection and treated with 200 nM rapamycin. After 2 d.p.f., the ESCO2-splice MO embryos looked more defective than those in which the ESCO2 mRNA was co-injected. (D). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-Splice MO (10 ng), and co-injected with a control mRNA or ESCO2 mRNA. 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. (E). Cranial development was quantified using the sum of the pq (palatoquadrate) cartilage and mc (Meckel′s cartilage) divided by cranial length. The measurement was done on 3 embryos per group. P<0.001, ESCO2-MO with Control-mRNA co-injection vs ESCO2-5mis with Control-mRNA co-injection; P<0.001, ESCO2-MO with ESCO2-mRNA co-injection vs ESCO2-MO with Control-mRNA co-injection. (F). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-Splice MO (10 ng), and immediately separated into egg water with L-Glutamine (L-Glu) alone (4 mM), L-Leu alone (10 mM), or L-Leu (10 mM) plus L-Glu (4 mM) treatment. After 3 d.p.f., optimal rescue was observed with L-Leu plus L-Glu. Scale bar = 200 μm. (G). Embryos (1–2 cells) were injected with ESCO2-5mis or ESCO2-Splice MO (10 ng), and immediately separated into egg water with D-His (10 mM), D-Leu (10 mM), L-Thr (10 mM) or L-Leu (10 mM) treatment. All treatments included L-Glu (4 mM). After 3 d.p.f., only embryos treated with L-Leu showed partial improvement of development. Scale bar = 200 μm.