Off-target Erk-KTR activity in the early zebrafish embryo

(A) Schematic of the Erk-KTR construct showing the N-terminal Erk-docking domain derived from ELK1, a nuclear localization sequence (NLS) containing Erk-consensus phosphorylation sites (P), a nuclear export sequence (NES) and a C-terminal fluorescent protein, Clover.

(B) Live images of an NIH-3T3 cell transfected with ubiP:Erk-KTR-Clover construct. The cytoplasmic-to-nuclear ratio of the Erk-KTR fluorescence provides a live readout of relative Erk activity levels. White-dashed line, cytoplasm; yellow-dashed line, nucleus.

(C) Combined immunofluorescence and RNAscope showing diphosphorylated Erk (P-Erk) and fgf8a expression relative to the dorsal organizer marked by goosecoid (gsc) expression. Embryos are oriented in an animal view (3.3 hpf) or lateral view (3.6-5.3 hpf). White-dashed line, embryo proper; gray-dashed line, yolk; 50% epi, 50% epiboly.

(D) Stills of live ubiP:Erk-KTR-Clover embryos. Embryos are false-colored to indicate Erk-KTR activity as readout by the KTR reporter in a binary manner (green, high activity; magenta, low activity). Embryos are shown from an animal-lateral view. Insets show a magnified view of the boxed region without false coloring.

(E) Schematic cross-section of the embryonic margin showing the relative position of the deep cells (DCs), the enveloping layer (EVL) and the yolk syncytial layer (YSL).

(F) Single z-slices showing Erk-KTR activity in the EVL and DCs from the indicated embryos in (D).

Scale bars, 25 μm (B), 50 μm (F), or 100 μm (C and D).

Erk-KTR reports on Erk and Cdk1 activity in early zebrafish embryos

(A) Illustration of the method used to report Erk-KTR activity in early zebrafish embryos (schematized below) by measuring mean fluorescence intensity in a region of the nucleus (magenta) and cytoplasm (cyan). The margin of the embryo exhibits high Fgf signaling, while the animal pole does not.

(B) Live imaging of ubiP:Erk-KTR-Clover transgenic embryos at either the margin or animally, as indicated in (A), following treatment with DMSO (control) or 10 μM PD-0325901 (MEKi) for an hour from 4.0 hpf.

(C) Quantification of Erk-KTR activity in (B) at the margin (***p = 0.0002) and animally(*p = 0.0207). n = 178–209 cells per condition from 5 embryos. Shown arethe single-cell readouts of Erk-KTR activity overlayed with the per embryo averages and the overall mean.

(D) As in (B) but following treatment with DMSO (control), 20 μM RO-3306 (CDK1i), or both 10 μM PD-0325901 and 20 μM RO-3306 (MEKi + CDK1i) for 1 h from 4.0 hpf.

(E) Quantification of Erk-KTR activity in (D) as in (C) for CDK1i (margin p = 0.3131; animal p < 0.0001) or both MEKi and CDK1i (margin p < 0.0001; animal p < 0.0001). n = 107–146 cells per condition from 3 (DMSO and MEKi + CDK1i) or 4 embryos (CDK1i) per condition.

Statistical tests were Student t test (C) or one-way ANOVA with Sidak’s multiple comparisons test (E).

Scale bars, 20 μm; **** p < 0.0001; ns, not significant.

A modified Erk-KTR reports ERK activity in mouse fibroblasts

(A) Amino acid sequences of the Erk-docking domain of Erk-KTR and modified Erk-KTR (modErk-KTR) highlighting the modifications (red) made to reduce off-target reporter activity, including the R>A substitution within the docking site and the additional FQFP Erk-docking site between the ELK fragment and the NLS.

(B) Quantification of ERK activity in NIH-3T3 cells transfected with the Erk-KTR or modErk-KTR constructs (n = 58 cells each, mean ± SD). Cells were serum-starved overnight and ERK was induced by the addition of 10% FBS. ERK activity was inhibited after 30 min with 10 μM PD-0325901 (MEKi).

(C) Representative images of reporter activity in (B).

(D) Quantification of ERK activity in NIH-3T3 cells transfected with the modErk-KTR construct after overnight serum starvation, followed by the addition of different concentrations of FBS. Individual cell traces and the mean (black line) are shown for 0% FBS (n = 32 cells), 2% FBS (n = 57 cells), and 10% FBS (n = 30 cells).

(E) Individual cell traces from (D).

Scale bars, 20 μM.

modErk-KTR specifically reports on Fgf/Erk activity in early zebrafish embryos

(A and B) Stills of live ubiP:Erk-KTR-Clover transgenic embryos. Embryos are false-colored to indicate Erk activity levels; (green, high activity; magenta, low activity). Embryos are shown from an animal (A) or lateral (B) view. Insets show a magnified view of the region within the black box without false coloring. White-dashed line, embryo proper.

(Ai) Single z-slices through the center of embryos in (A) showing Erk activity around the embryonic margin using the same color scheme as (A).

(C) Live imaging of ubiP:Erk-KTR-Clover transgenic embryos following treatment with DMSO (control), 10 μM PD-0325901 (MEKi), 20 μM RO-3306 (CDK1i), or both MEKi and CDK1i for an hour from 4.0 hpf.

(D) Quantification of Erk activity in (C) at the margin and animally. Shown are the single-cell readouts of Erk activity overlayed with the per embryo averages and the overall mean for DMSO (control, n = 209 cells from 5 embryos [margin] or n = 200 cells from 5 embryos [animal]), 10 μM PD-0325901 (MEKi; n = 137 cells from 4 embryos [margin] or n = 196 from 5 embryos [animal]), 20 μM RO-3306 (CDK1i; n = 227 cells from 6 embryos [margin] or n = 133 cells from 4 embryos [animal]) or both 10 μM PD-0325901 and 20 μM RO-3306 (MEKi + CDK1i; n = 183 cells from 5 embryos [margin] or n = 194 cells from 5 embryos [animal]) treated embryos.

(E) Live imaging as in (C) of embryos injected with either 25 pg fgf8a or 500 pg dnFGFR at one-cell stage. Embryos were imaged at 50% epiboly (5.3 hpf).

(F) Quantification of Erk activity in (E) as in (D) for control (n = 142 cells from 3 embryos [margin] and n = 116 cells from 3 embryos [animal]), fgf8a (n = 130 cells from 4 embryos [margin], or n = 192 cells from 4 embryos [animal]) or dnFGFR (n = 168 cells from 4 embryos [margin] or n = 157 cells from 4 embryos [animal]) injected embryos.

Statistical tests were one-way ANOVA with Šidák’s multiple comparisons test.

Scale bars, 100 μm (A and B), 50 μm (Ai) or 20 μm (C–E); **** p > 0.0001.

See also Figure S1.

Improved Erk activity reporting with modERK-KTR in Drosophila embryos and larval tissue

(A) Schematic of anteroposterior Torso/ERK signaling during early Drosophila development where signaling is restricted to both poles of the blastoderm (shown by orange gradient), excluding the pole cells (cluster of green cells on the right-hand side). Cells shown with black nuclei have high ERK signaling, whereas those with green nuclei have no ERK signaling.

(B and C) Representative images of the posterior half of transgenic Drosophila embryos maternally expressing the original ERK-KTR (B) or modERK-KTR (C) constructs with a polycistronic H2Av-mCherry tag during cell cycles (cc) 13 and 14. Shown is a single z-slice through the center of the embryo (top) and a magnified view of the regions indicated by white boxes (bottom).

(D and E) Representative images of eye imaginal discs ubiquitously expressing ERK-KTR (D) or modERK-KTR (E) constructs with a polycistronic H2Av-mCherry tag under the control of tub-Gal4. The levels of ERK activity, as read out by ERK-KTR constructs, are here compared with the levels of P-ERK.

(F) Quantification of (D) and (E) comparing P-ERK levels with the read out of ERK-KTR (n = 222 cells from 3 discs) or modERK-KTR (n = 293 cells from 3 discs) constructs and fitted with a simple linear regression.

See also Figures S3 and S4.

modERK-KTR reads out Fgf/Erk signaling gradient formation in real time

(A) Schematic of the zebrafish embryo illustrating the lateral region imaged in (B) relative to the dorsal organizer (see Figure 1C).

(B) Quantification of Erk activity (log₂(C/N)) in the lateral region of ubiP:modErk-KTR-Clover embryos at 20 min intervals from dome (4.3 hpf) to germ ring stage (5.6 hpf). Cells were binned based on their distance in cell tiers from the embryonic margin (0). n = 3 embryos showing the per embryo mean ± SD. Also shown is an overlay of the mean levels at each time point.

(C) Overlay of the mean Erk activity (modErk-KTR) and P-Erk levels in similarly staged embryos (5.3 hpf).

(D) Representative immunofluorescence images of embryos treated with DMSO (control) or MEKi (10 μM PD-0325901) for 10–20 min from 5.0 hpf before fixation.

(E) Quantification of P-Erk levels from (D) in cell tiers relative to the embryo margin (0) showing mean ± SD. DMSO 10 min, 5 embryos; MEKi 10 min, n = 6 embryos; DMSO 20 min, n = 4 embryos; MEKi 20 min, n = 4 embryos.

(F) Quantification of modErk-KTR read out following treatment with DMSO (control) or 10 μM PD-0325901 (MEKi). Shown is the mean of n = 3 embryos per time point.

(G) Schematic comparing the dephosphorylation rates of Erk and its targets.

Scale bars, 50 μm.

See also Figure S5.

Mitotic erasure induces oscillatory Fgf/Erk signaling dynamics in the presumptive mesendoderm

(A) Representative images of a single mesendodermal cell approaching mitosis. ubiP:modErk-KTR-Clover embryos were injected with 25 pg H2B-mScarlet-I mRNA at the one cell stage and a lateral region of the margin was imaged from ~4.6 hpf at 1 min intervals. White-dashed line labels the single cell.

(B) As in (A) following two cells post-mitosis. Black line labels the nucleus.

(C) Quantification of Erk activityfrom (A) and (B) following mother cells (n = 56 cells) from −5 min before mitosis and daughter cells (n = 110) +45 min after mitosis showing mean ± SD. Nuclear envelope breakdown means the KTR cannot read out Erk activity during mitosis itself.

(D) Representative immunofluorescence images of P-Erk and P-H3 in zebrafish embryos (4.6 hpf).

(E) Quantification of the time to Erk reactivation (log₂(C/N) ≥ 0.25) post-mitosis and the distance of each cell from the embryonic margin (n = 110) and fitted with a simple linear regression.

(F) Comparison of Erk reactivation rates from (C) with cells binned based on their initial distance from the margin.

(G) Schematic of the Erk activity gradient, as read out by modErk-KTR, and the extracellular levels of Fgf8a-GFP described in similarly staged embryos (~5.3 hpf).⁴³

(H) Single-cell traces of sister cells post-mitosis from (E).

(I) Model depicting how mitotic erasure of P-Erk and its target proteins induces signaling oscillations. Both the rate of reactivation post-mitosis and the final amplitude of Erk activity are sensitive to a cell’s relative position within the Fgf signaling gradient. Coupled with cell cycle asynchrony and variability in reactivation rates, mitotic erasure introduces heterogeneity to Fgf/Erk signaling in the presumptive mesendoderm. Different green lines correspond to P-Erk levels in different cells.

Scale bars, 10 μm.

See also Figures S6 and S7.