Fig. 4

Fig. 4

Increased ERK signal measured by spectral unmixing and AB-FRET in Shp2^D61G zebrafish mutants showing morphological defects. (A) Representative bright-field micrographs and graphs showing body length measurements of embryos overexpressing the WT and mutant (D61G) form of Shp2 at 55 hpf. Non-parametric Mann Whitney test is used to assess the statistical significance (*** p < 0.001). N = 28 and 23 (Shp2^WT and Shp2^D61G respectively). Data are expressed as median with interquartile range. (B) Representative bright-field micrographs of mutant embryos at early segmentation stage ( 11 hpf) compared to control fish (expressing Shp2^WT). Major and minor axes defects are visible, outlined by a dashed orange lines and by the quantification of major/minor axis ratio. The box plot with the median (middle line), 25th–75th percentiles (box), and min–max values (whiskers) shows the quantification of major/minor axis ratios. One-tail Student t-test is used to assess the statistical significance (**** p < 0.0001). N = 77 and 64 (Shp2WT and Shp2D61G respectively). (C, D) Single plane images of confocal x,y,z,λ,t and sum-intensity projections of confocal x,y,λ,z live scans (C and D, respectively) obtained by spectral unmixing-FRET of embryos at 11 hpf (C) and 5 hpf (D) expressing Shp2^WT and Shp2^D61G and showing FRET (red) and CFP (donor, green) signals. Increase in ERK activity (FRET channel) in the tail bud (PSM) (C) and in the margin of the animal pole (D) of zebrafish embryos is indicated by white arrows. A dashed white line outlines the developing embryo (C) and the margin (D). Close-ups on the right rendered with “Smart” LUT in Fiji show increased signal in the tail (C) and margin (D) regions. (D’, D’’) Bar graphs reporting the quantification and statistical support for FRET/CFP ratio (raw integrated density, arbitrary units, a.u. defined as “FRET index”) and expressed as raw values (D’) as fold change (FC) of Shp2^D61Gvs. Shp2^WT (D’’). Data are expressed as mean ± SEM. One-tail t-test is used to assess the statistical significance (* p < 0.05, *** p < 0.001)). N of embryos = 4 (Shp2WT and Shp2D61G). (E) Representative confocal images (single plane) showing donor (CFP, green) before (Donor-Pre) and after (Donor– Post) AB-FRET for embryos expressing Shp2^WT and mutants expressing Shp2^D61G. A dashed white line indicates the animal pole margin targeted for Acceptor Bleaching (AB). For each condition, insets of the right show close-ups on the donor (CFP) at the margin region before and after AB (pre- and post-, respectively) rendered with “Smart” LUT in Fiji. Bright pixels showing FRET signal increase upon AB are indicated by white arrows. Embryos are outlined by a continuous white line. (E’-E’’) Box plot with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers) showing the quantification of AB-FRET efficiency (E %, E’) and RDA values (nm, E’’) in the margin of zebrafish 5hpf embryos overexpressing WT and mutant (D61G) Shp2. One-tailed t-test is used to assess the statistical significance (* p < 0.05, ** p < 0.01). N = 8 and 5 (Shp2WT and Shp2D61G respectively). L1-L3: different analysis levels for both morphological and molecular assessments. Source data are provided as a Source Data file.