Fig. 2

ERK activity is high in proliferative cells of the periderm. (A) Stills from transgenic embryos expressing krt4:H2A-mCherry and krt4:venus-hGeminin, treated with DMSO or 2 µM PD0325901 for 18 h. Embryos treated with PD03 have reduced cycling cells seen by a decrease in geminin-positive cells. (Scale bar, 100 µm.) (B) Quantification of the fraction of geminin-positive cells in embryos treated with DMSO or PD0325901 shows that PD03-treated embryos have significantly decreased cycling cells seen by reduction in normalized fraction of geminin-positive cells. Average of DMSO = 1, n = 13 embryos, Average PD03 = 0.5455, n = 13 embryos. P = 0.0012. (C) The ERK translocation sensor shuttles between the nucleus and cytoplasm upon phosphorylation, with the cytoplasmic-to-nuclear signal ratio indicating inferred ERK activity in single cells. (D) Examples of high, intermediate, and low ERK activity in periderm cells. The sensor shuttles into the nucleus as ERK activity decreases. (Scale bar, 10 µm.) (E) Snapshot of embryos expressing krt4:ERK-KTR-mCerulean and the corresponding heat map of ERK activity. ERK activity is high in most cells of the periderm without any specific spatial pattern at 28hpf. (Scale bar, 100 µm.) (F) Quantification of ERK activity levels in periderm cells. Mitotic cells (blue) have higher ERK activity compared to nonmitotic cells (red), in the hours leading to division. Cells were classified as mitotic if they divide during the imaging. T = 0 is nuclear division. (G) Stills from timelapse of mitotic and nonmitotic cells showing higher ERK activity in mitotic cells. Arrowheads point to the mitotic cell and nonmitotic cell in the Upper and Lower panels, respectively. (Scale bar, 10 µm.) (H) Average ERK activity 1 to 5 h before mitosis is significantly higher than that compared with random 4 h window for nonmitotic cells. Median of average ERK activity for mitotic cells – 1.054, N = 40 cells and nonmitotic cells – 0.734, N = 75 cells, t test P = 2.3852e-18.