Fig. 5

Reduced ERK activity measured by spectral unmixing and AB-FRET in zebrafish Shp2^D61G NS-causing mutants exhibiting morphological defects upon low- and high-dose MEK inhibitor treatment. (A) Representative bright-field micrographs of hatched zebrafish embryos expressing Shp2^WT, Shp2^D61G treated with DMSO vehicle control (Shp2^D61G) or with 0.25 µM and 1 µM PD0325901 (PD) since 4 hpf. The bar graph on the right shows body length measurements of zebrafish embryos expressing Shp2^D61G and the rescue obtained by low-dose and high-dose PD0325901 treatment. One-way ANOVA with Dunnett’s (^a 0.06, **** p < 0.0001) and Holm-Sidak (only for low-dose PD0325901 experimental group, ^b * p < 0.05) post hoc test is used to assess statistical significance after outliers’ removal (ROUT method Q = 1%). N = 31, 50, 43 and 44 (Shp2^WT, Shp2^D61G - or + 0.25 µM PD and 1 µM PD respectively). (B) Representative bright-field micrographs of mutant embryos at early segmentation stage treated either with DMSO vehicle control (Shp2^D61G) or with 0.25 µM and 1 µM PD compared to control fish (expressing Shp2^WT). Major and minor axes defects are visible, outlined by a dashed red line, embryos are outlined by a dashed black line. Quantification of major/minor axis ratio is shown by the box plot with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers). One-way ANOVA with Dunnett’s (^a 0.051, * p < 0.05, **** p < 0.0001) and Holm-Sidak (only for low-dose PD0325901 experimental group, ^b * p < 0.05) post hoc test are used to assess the statistical significance after outliers’ removal (ROUT method Q = 1%). Data are expressed as mean ± SEM of two independent biological replicates. N = 27, 32, 25 and 19 (Shp2^WT, Shp2^D61G - or + 0.25 µM and 1 µM PD, respectively). (C) Representative confocal images (single plane) showing donor (CFP, green) before (Donor-Pre) and after (Donor–Post) AB-FRET for mutants treated with DMSO vehicle control (Shp2^D61G) or with the PD0325901 (Shp2^D61G + 0.25 µM and 1 µM PD). A dashed white line indicates the animal pole margin targeted for Acceptor Bleaching (AB). For each condition, insets on the right show close ups on the donor (CFP) at the margin region before and after AB (pre- and post-) rendered with “Smart” LUT in Fiji. The acceptor (YPet, blue) is shown in the small inset below before and after bleaching of the margin region (dashed white line and arrow). Embryos are outlined by a continuous white line. (D) Quantification of signal intensity before AB-FRET. The upper and lower scatter plots (Median and interquartile range) show Acceptor/Donor (YPet, CFP) and CFP signal intensity, respectively. N = 18, 9 and 12 (shp2^D61G, Shp2^D61G + 0.25 µM PD and Shp2^D61G + 1 µM, respectively). A marked reduction in the FRET (and reduced Donor quenching) is observed in NS mutants treated with 1 µM PD. Kruskal-Wallis with Dunn’s post hoc test is used to assess statistical significance (* p < 0.05). Data are expressed as median with interquartile range. N of embryos = 18, 9 and 12 (Shp2^D61G, Shp2^D61G + 0.25 µM PD and Shp2^D61G + 1 µM, respectively). (D’) AB-FRET data quantification represented by the box plot with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers) and showing the AB-FRET efficiency (E %) and RDA values (nm, right inset) in the margin of mutant embryos (Shp2^D61G) treated with DMSO vehicle control or low and high-dose of PD0325901 (Shp2^D61G + 0.25 µM PD or 1 µM PD, respectively). One-way ANOVA with Dunnett’s post hoc test is used to assess the statistical significance (* p < 0.05). For FRET efficiency (E) dataset, n = 18, 9 and 12 (Shp2^D61G, Shp2^D61G + 0.25 µM PD and Shp2^D61G + 1 µM, respectively). For R_DA dataset, values are excluded when E % values = 0 (exclusion criterion reported in the source data file), N = 17, 9 and 8 (Shp2^D61G, Shp2^D61G + 0.25 µM PD and Shp2^D61G + 1 µM, respectively). The lower graph shows the percentage of embryos classified based on high or low values of AB-FRET-derived E (> or < 7.53, respectively). One-sided Chi-square’s test in a 2 × 2 contingency table (shp2^D61G vs. Shp2^D61G + 0.25 µM PD ns = not statistically significant, Shp2^D61G vs. Shp2^D61G + 1 µM PD * p < 0.05) is used to assess statistical significance. N = 18, 9, and 12 (Shp2^D61G, Shp2^D61G + 0.25 µM PD and Shp2^D61G + 1 µM, respectively). L1-L3: different analysis levels (L1 and L2, morphological; L3 molecular). Source data are provided as a Source Data file