Fig. 2

Increase in ERK activity is observed by spectral unmixing-FRET in the forebrain of live Teen embryos upon EGF ventricle injection. (A) The schematics depicts the experimental approach used to stimulate local pERK increase (ERK activity) within the anterior brain upon injection of rat EGF within the forebrain ventricle in live 24 hpf fish. FRET imaging using spectral unmixing mode was performed before (T0) and after T2 (30’) injection (T1). The lower schematics indicates the FRET signal analysis performed in various regions of interest around the ventricle, as indicated in the legend. (B) Representative sum-intensity projections of confocal x,y,z, λ live scans obtained by spectral unmixing from two embryos showing signal relative to FRET (red) and CFP (donor, green) before (pre) and 30 min after EGF injection (pre and post EGF, respectively). Red arrows mark FRET signal increase. D: dorsal, V: ventral, ML: medio-lateral domains. (C) Close ups of the sum-intensity projections of superficial z-layers of the ratiometric image (FRET/CFP) rendered with the “smart” LUT intensity scale and showing high FRET signal correlating to ERK activity (white arrows) in the dorsal (D), mediolateral (ML) and ventral (V) domains. (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 fold change (FC) of treated vs. control fish. Dorsal: quantification in the dorsal domain. Mediolateral + Ventral: FC of the intensity detected in the medio-lateral (mean between right and left side) + the intensity of the ventral domain. Fold change (FC) data are expressed as mean ± SEM of two independent biological replicates, n = 2 embryos. Paired one-tailed t-test is used to assess statistical significance (* p < 0.05). Source data are provided as a Source Data file.