Ranking of MEK1 RASopathy mutations in the zebrafish heart. (A) Schematic of zebrafish orientation during heart imaging. (B) Representative images of the zebrafish heart at 19.5 hpf with the outline of the heart in yellow. Cardiac cells are labeled by membrane GFP expressed from the myl7 promoter. (Scale bar, 50 ?m.) (C) Rank of selected mutations based on zebrafish heart size in embryos overexpressing MEK variants (+) compared with uninjected siblings (?, black). The average for each mutation is plotted with the SEM indicated. RASopathies, blue; RASopathies and cancer, green; cancer only, red. Here, N values are N_E203Q,? = 12, N_E203Q,+ = 9, N_G128V,? = 12, N_G128V,+ = 9, N_F53L,? = 9, and N_F53L,+ = 8. One-way analysis of variance (ANOVA) with Bonferroni correction was used for statistical analysis: *P < 0.1. The differences of the following pairs were not statistically significant: E203Q, G128V; G128V, F53L.

Measurement of oval shape dimensions of the zebrafish embryo at 11 hpf. Prior to imaging, each embryo was manually positioned so that it was lying laterally. Embryos that died or developed improperly were not considered for measurement. The major (a) and minor (b) axes of the yolk of the embryo were measured on ImageJ, using the Measure feature after drawing a line between the two points demarcating the start and the stop of the axis. The line for measurements were drawn so that the maximum length between the two ends is captured. An example (A) WT-MEK injected embryo, (B) G128V-MEK injected embryo, and (C) F53L-MEK injected embryo are shown.