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Fig. 3
Genetic interaction of ccm2l with ccm1 and ccm2 and modulation of mekk3 activity in zebrafish. (A) Single-cell flk-GFP embryos were injected with control (ctrl) or ccm2l MO (MO-1), and images were obtained at 48 h postfertilization. The arrow indicates cardiac enlargement/big heart. (B) The big heart phenotype was evaluated with increasing doses of ccm2l MO-1. (C–E) Different amounts of ccm2l MO-1 or ctrl MO were injected into embryos from san/ccm1 (C) and vtn/ccm2 (D) heterozygous crosses. The vtn/ccm2 zebrafish with ccm2l knockdown show further enlargement of the heart in combination with heart failure (HF) and tail/axis defects (arrows). Phenotypes were classified as WT (no phenotype), big heart, HF/axis (HF and axis/tail defects), or dead (lack of survival). (E) Representative pictures of vtn/ccm2 mutants without (Left) or with ccm2l MO-1 injected (Right). (F) Rescue of the axis phenotype in ccm2/ccm2l-deficient fish with 4 ng/µL mekk3 MO. Embryos from vtn/ccm2 heterozygous crosses were injected with ccm2l alone or in combination with ctrl or mekk3 MO, and the axis defect phenotype was scored. (G) Rescue of the big heart phenotype in vtn/ccm2 mutant fish (vtn) with ctrl or 2 ng/µL mekk3 MO. (H) Rescue of big heart in WT plus 4 ng/µL ccm2l MO-1 injection with ctrl MO or indicated concentrations of mekk3 MO. (I and J) Rescue of big heart (I) and tail axis defects (J) in vtn + ccm2l MO-1 zebrafish, with human CCM2L mRNA. The big heart phenotype in vtn alone (I, ) is shown for comparison. *P < 0.05, unpaired t test. Graphs represent data pooled from three independent experiments. Error bars represent SEM.