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Fig. 1
Characterization of ubi:ERK-KTR-Clover in zebrafish embryos. (A) Schematic illustration of the DREKA transgenesis vector pDESTubi:ERK-KTR-CloverpATol2. (Tol2, Tol2 recombinase recognition sites; ubiquitin, ubiquitin promoter; Elk1312−356, ERK docking sites from Elk1; bNLS, bipartite nuclear localization signal; P-sites, phosphorylation sites; NES, nuclear export signal; modified after Regot et al., 2014) (B) Schematic depiction of the ERK-KTR reporter principle. ERK signaling inactive: reporter is mainly localized in the nucleus; ERK signaling active: reporter is localized in the cytoplasm sparing the nucleus. (C) pDESTubi:ERK-KTR-CloverpATol2 and H2B-CFP mRNA co-injected wildtype zebrafish embryos express ERK-KTR-Clover in a mosaic manner. mClover fluorescence is mainly found in the cytoplasm of skin epithelial cells, indicating active Erk signaling (upper panel) and in the nucleus of muscle cells indicating absence of Erk signaling activity (lower panel). (C′) Quantification of ERK-KTR localization in skin epithelial cells and muscle cells at 48 hpf. Green, mClover fluorescence in the nucleus; Red, mClover fluorescence distributed throughout the cell; Blue, mClover fluorescence in the cytoplasm sparing the nucleus (n = 385 cells, 10 embryos at 48 hpf). (D) Skin epithelial cells of co-injected embryos incubated with MEK1/2 inhibitor PD98059 (30 μM) overnight show mClover fluorescence in the nucleus at 48 hpf indicating the reporter responds to Mek inhibition. (D′) Quantification of mClover localization shows reporter shuttling to the nucleus after MEK1/2 inhibition (n = 300 cells, 5 embryos at 48 hpf). (E) Muscle cells expressing constitutively active HRAS (zebrafish injected with pDESTubi:ERK-KTR-CloverpATol2, H2B-CFP:UAS:HRASG12V, and KalTA4 mRNA Distel et al., 2009) show active Erk signaling at 48 hpf. (E′) Quantification of mClover localization shows constitutively active HRAS induced reporter shuttling to the cytoplasm (n = 235 cells, 5 embryos at 48 hpf). (F) Mitotic skin epithelial cells of pDESTubi:ERK-KTR-CloverpATol2 injected zebrafish at 24 hpf. Dividing skin epithelial cells (white arrowheads) show dynamic Erk signaling with a sudden change from cytoplasmic to nuclear reporter localization before cytokinesis [compare time points 11:24 min/13:41 min (left arrowhead) and 13:41 min/21:40 min (right arrowhead)]. After cytokinesis the reporter remains in the nuclei of both daughter cells (arrowheads at 1:00:28 h). Images taken from a time-lapse movie (Movie 2) are maximum projections of several planes. All scale bars are 25 μm. All images were recorded on a Leica SP8 X WLL confocal microscope and rendered using Photoshop CS6.