Figure 1. 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).