(related to Fig. 6). Knockdown of zC2orf62 results in notochord undulation and ciliogenesis defects in the olfactory organ. (A) In situ hybridization of ntla and shha at 24 hpf shows that more than 90% of C2orf62_1 MO-injected embryos have an undulated notochord, in contrast to controls (lateral views, scale bars, 200 μm). (B) Embryos were either not injected (n.i.) or injected with 5 or 10 ng C2orf62_1 MO and observed at 48 hpf. C2orf62_1 MO-injected embryos display a curved body, heart edema (arrow), and a small head with incorrectly defined brain structures, small eyes and small ears (dashed circle). Scale bars, 200 μm. (C) Embryos were injected either with 2.5 ng Ctrl. MO or C2orf62_1 MO, and ciliated cells from the olfactory organ (OO) were visualized using anti α-acetylated tubulin at 48 hpf. The olfactory organs of C2orf62_1 MO-injected embryos are smaller and display less ciliated cells than controls. Scale bars, 50 μm. (D) Embryos were injected with 850 pg mRNA coding for the C-terminal part (aa 276-356) of zC2orf62,either wild type (WT C-ter) or alanine-mutated on Leu-318 and Phe-341 (L318A/F341A C-ter), and observed 24 h later. Phenotypic traits 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 graph shows the percentage of embryos in each category. The percentage of normal (white) embryos is significantly higher for L318A/F341A C-ter mRNA-injected embryos than for WT C-ter mRNA-injected embryos (25.7% for WT C-ter mRNA and 80.6% for L318A/F341A C-ter mRNA P = 0.0078, Student′s t test).
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