Fig. S5

(related to. Fig. 6). Evaluation of the specificity of C2orf62 morpholinos. Zebrafish embryos were injected with different quantities of C2orf62_1 or C2orf62_2 MO and observed under a stereomicroscope. (A-C) Phenotypic traits at 24 hpf were classified into four categories, each being associated with one grey code, as reported in Fig. 6A: normal (white, indistinguishable from controls), severe (clear grey, defect of brain structure formation and short body axis), very severe (dark grey, indistinguishable left/right or front/back polarity), and lethal (black). The bar graphs show the percentage of embryos in each category. (A) Although the injection of 1.25 ng C2orf62_1 or C2orf62_2 MO has no effect on embryo morphology, the co-injection of 1.25 ng C2orf62_1 MO and 1.25 ng C2orf62_2 MO results in 47% of severe-to-lethal phenotypes, showing a strong synergy between both MOs. (B) Embryos were injected with 5 or 10 ng C2orf62_2 MO together with 0 (buffer alone), 15 or 30 ng P53 MO. Coinjection of P53 MO does not significantly change the phenotypes induced by injection of C2orf62_2 MO alone. (C) Embryos were injected with 10 ng C2orf62_2 MO and/or 2 ng full-length zC2orf62 mRNA. The percentage of normal (white) C2orf62_2 MO-injected embryos is significantly higher in the presence of mRNA (1.96% for C2orf62_2 MO alone and 12.53% for C2orf62_2 MO + RNA, P = 0.0044, Student′s t test). (D) Embryos were co-injected with 680 pg C2orf62-EGFP mRNA and 5 ng Ctrl. MO, C2orf62_1 MO or C2orf62_2 MO and observed 6 h later under a fluorescence stereomicroscope. EGFP signal was abrogated in all C2orf62_1 MO-injected embryos (star marked), and unexpectedly in 33% of C2orf62_2 MO-injected embryos. (E). RT-PCR analysis of total RNA extracted from 24 hpf embryos after injection of 5 ng of Ctrl. MO or C2orf62_2 MO shows that C2orf62_2 MO alters zC2orf62 splicing.