Fig. 6

Molecular and morphological effect of the treatment with MEK inhibitor PD0325901 on zebrafish embryos overexpressing Shp2^D61G and showing developmental features modeling NS. (A) AB-FRET efficiency (E %, x) and RDA values (nm, y) calculated from AB-FRET on 5 hpf zebrafish embryos expressing mutant (D61G, red) Shp2 - and + treatment with low (0.25 µM, light green) and high (1 µM, dark green) doses of the MEK inhibitor PD0325901. High E values correlating with low R_DA values are schematically indicated by a red bar as the “genetic effect” relative to the Shp2^D61G allele. On the contrary low E values correlating with high R_DA values are indicated by a green bar as the “treatment effect” on the NS mutants due to short PD exposure. N of embryos = 18, 9, 12 (Shp2^D61G, Shp2^D61G + 0.25 µM PD and Shp2^D61G + 1 µM, respectively). (B) Schematics and x,y graph showing the correlation between ERK activation (measured as AB-FRET E FC, x axis) and body axes (measured as ratio between major and minor axis, y axis) in zebrafish embryos expressing Shp2^D61G compared to control (Shp2^WT) (left) or Shp2^D61G treated with DMSO vehicle control or with low- and high- PD doses (right). Modulations of the oval shape of the embryo measured at 11 (y axis) and of ERK activity measured by FRET already at 5 hpf (x axis) are depicted by a schematic illustration. A Heat map on the side of the graph indicates the severity of body shortening at 55hpf (dark bronze = severe). (C) Summary heat maps showing the correlation between ERK activity at 5hpf (orange, bottom graph, obtained by AB-FRET measurement), body axes morphology at 11hpf (violet, middle graph, major/minor axis ratio measurement) and embryo elongation at 55hpf (bronze, upper graph, body length measurement) in NS mutants expressing Shp2^D61G treated with DMSO vehicle control or with low (0.25 µM) and high (1 µM) PD0325901 doses. High ERK activity in NS mutants (expressing Shp2^D61G) decreases in a dose-dependent manner upon short-time treatment with low and high PD0325901 doses. Accordingly, in a dose-dependent effect partial or complete morphological correction is observed at the level of body axis measured at 11 hpf. Toxicity with prolonged 1 µM PD0325901 treatment is shown for body length (worsening of the body length phenotype measured in embryos at 55 hpf)