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First published online 5 January 2005
doi: 10.1242/dev.01607

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ERK1 activation is required for S-phase onset and cell cycle progression after fertilization in sea urchin embryos

Rada Philipova^*, Jolanta Kisielewska^*, Pin Lu, Mark Larman, Jun-Yong Huang and Michael Whitaker

University of Newcastle upon Tyne, Institute of Cell and Molecular Biosciences, Medical School, Framlington Place, Newcastle NE2 4HH, UK

View larger version (99K): [in a new window]   Fig. 4. XCL100 and U0126 inhibit decondensation of sperm chromatin after pronuclear fusion. Hoechst 33342 dye DNA staining of control embryos, embryos microinjected with 2.5 µM recombinant XCL100 and embryos incubated with 100 µM U0126. The times indicated in the Control panels apply to all three conditions. Arrows indicate sperm chromatin within zygote nucleus. Inset at 120 minutes shows the control embryo undergoing cytokinesis, while the XCL100- and U0126-treated embryos have not undergone NEB. Scale bar: 50 µm.

View larger version (38K): [in a new window]   Fig. 2. XCL100 phosphatase prevents both sperm chromatin decondensation in early embryos and nuclear accumulation of GFP-PCNA in embryos and Ca2+ ionophore-activated eggs. (A) Wide-field fluorescence microscopy of embryos microinjected with GFP-PCNA (control) and, in addition, with XCL100 (1.5 µM and 2.5 µM final concentration) before fertilization and imaged at 65 minutes after fertilization, just before NEB. XCL100 (2.5µM) blocked the cell cycle and NEB did not subsequently occur. Arrow indicates uncondensed sperm chromatin. (B) Nuclear GFP-PCNA fluorescence intensity (mean±s.e.m., control, n=6; 1.5 µM XCL100, n=3; 2.5 µM XCL100, n=4; 2.5 µM XCL100 unfertilized eggs, n=3) measured during the course of the cell cycle in the experiments shown in A. Five different females were used in experiments with embryos (graphs and bars) and three females in the experiments with unfertilized control eggs. Inset shows that 2.5 µM XCL100 prevented NEB. (C) Confocal images of nuclear GFP-PCNA in control (n=3) and XCL100-microinjected eggs (n=4) activated with A23187 (20 µM). The time-course of GFP-PCNA fluorescence intensity in the nucleus is also shown. NEB was not observed in the presence of XCL100. Scale bar: 50 µm.

View larger version (41K): [in a new window]   Fig. 3. U0126 inhibits nuclear accumulation of GFP-PCNA, activation of ERK1, and DNA synthesis in early embryos (A-F) and also in ionophore-activated eggs (G-H). (A) Nuclear accumulation of GFP-PCNA and subsequently NEB are prevented by 100 µM U0126, but not by 20 µM U0126 or 100 µM U0124 in early embryos. Times after fertilization are indicated. Scale bar: 50 µm. (B) Intensity of GFP-PCNA nuclear fluorescence in early embryos treated as in A and, in addition, in unfertilized eggs incubated in 20 µM and 100 µM U0126. Each graph represents the mean fluorescence value from three to five different experiments; in each experiment different females were used. (C) DNA synthesis is prevented by 100 µM U0126 in embryos. Incorporation of [3H]-thymidine in embryos (control, white circle; treated with U0126, black circle). (D) U0126 alone does not trigger DNA replication in unfertilized eggs (black circle), but treatment with the Ca2+ ionophore A23187 (20 µM) as a control does induce DNA synthesis (white circle). (E) U0126 (100 µM) applied 10 minutes before fertilization abolishes the post-fertilization peak in ERK1 activity. We measured MAP kinase activity in cell extracts during the first 8 minutes after fertilization: control, black circle; after U0126 treatment, black square. (F) Western blotting of immunoprecipitated active MAP kinase dimers, detected with anti-dual phosphorylated MAP kinase antibody (New England Biolabs). Control, 0 and 6 minutes; 100 µM U0126 treated (+U0126), 0 and 6 minutes; control immunoprecipitation in the absence of antibody (-AB, at 6 minutes). (G) Wide-field fluorescence images and intensity of nuclear fluorescence of unfertilized eggs microinjected with GFP-PCNA, incubated in 100 µM U0126 and activated with Ca2+ ionophore (A23187, 20 µM), together with controls. Nuclear fluorescence was not observed in eggs treated with U0126. Scale bar: 50 µm. (H) Incorporation of [3H]-thymidine in unfertilized eggs treated with 20 µM Ca2+ ionophore A23187. Ionophore activated eggs (white circle); eggs treated with U0126 15 minutes before ionophore activation (black circle).

View larger version (70K): [in a new window]   Fig. 1. GFP-PCNA accumulates in the embryonic nucleus during S phase through an aphidicolin-dependent process. (A) mRNA encoding GFP-PCNA (green, final concentration 0.0013 µg/µl), together with rhodamine-labelled tubulin (red, final conc. 0.01 µg/µl) were microinjected 100 minutes before two-channel confocal image recording. Time after fertilization is indicated. Second cell cycle, 101-133 minutes; S phase of the third cell cycle, 134-139 minutes. Scale bar: 50 µm. (B) GFP-PCNA protein was microinjected before fertilization. Confocal images of the nuclei at various times during the first cell cycle of control embryos (upper panel; scale bar, 15 µm) and embryos treated with aphidicolin (20 µg/ml at 15 minutes pre-fertilization: lower panel; scale bar, 100 µm) are shown. The measured average intensity of fluorescence of the female nucleus throughout the first cell cycle in both conditions is also shown. The time of each image corresponds to the time indicated on the graph. Aphidicolin caused a cell cycle block and NEB was not observed. The graph shows mean±s.e.m. from six control experiments and from three experiments with aphidicolin treatment. For each experiment a different female was used.

View larger version (41K): [in a new window]   Fig. 5. Effects of PD98059 on nuclear accumulation of GFP-PCNA in both unfertilized and fertilized eggs. GFP-PCNA fusion protein was microinjected into sea urchin eggs and the fluorescence intensity was observed in the presence of MEK inhibitor PD98059. (A) Confocal images of GFP-PCNA nuclear fluorescence in 20 µM PD98059-treated and control eggs. Scale bar: 50 µm. (B) Intensity of nuclear GFP-PCNA fluorescence in unfertilized eggs treated with 2. 5µM and 20 µM PD98059, and with 20 µM PD98059 in the presence of aphidicolin (20 µg/ml 15 minutes before addition of PD98059). (C) [3H]-Thymidine incorporation in unfertilized eggs treated with ionophore (Control) or 20 µM PD98059. Representative of three independent experiments. (D) PD98059 (20 µM) does not inhibit the nuclear accumulation of GFP-PCNA in embryos after fertilization. Each graph in B and D represents mean±s.e.m. from three or five experiments; in each experiment different females were used.