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First published online 4 July 2007
doi: 10.1242/dev.003756

Development 134, 2751-2759 (2007)
Published by The Company of Biologists 2007
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Requirement for ERK MAP kinase in mouse preimplantation development

Momoko Maekawa1,*, Takuya Yamamoto1,*, Michiaki Kohno2, Masatoshi Takeichi3 and Eisuke Nishida1,{dagger}

1 Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
2 Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14, Bunkyomachi, Nagasaki 852-8521, Japan.
3 RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047, Japan.


Figure 1
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  		Fig. 1. Effect of U0126 and PD184352 on preimplantation development. (A) Schedule of mouse preimplantation development (top). At the time points indicated, embryos were treated with inhibitors (Drug, arrows) and observed (obs., arrowheads). The inhibitor was added at the late two-cell stage, and embryos were examined 1 day (2.5 dpc) and 2 days (3.5 dpc) later. The zona pellucida was removed before inhibitor treatment. (Below) Embryos treated with 20 µM U0126 or 5 µM PD184352 were compared with control embryos (control, DMSO and 20 µM U0124). Typical images of embryos are shown. The bottom row shows the results when U0126 treatment was allowed to continue for 24 hours but the drug was then washed out. Observation of these embryos took place at 2 and 3 days after initial treatment. (B) Reversibility of the inhibitor-induced developmental arrest. (Bottom right) Late two-cell-stage embryos shown in the bottom row were treated with 20 µM U0126 for 24 hours before the U0126 was washed out. The embryos were cultured in a drug-free medium and then observed until E4.5. The top two rows show control embryos, which were treated with DMSO (vehicle, top) or U0126 that was not washed out (middle). dpc, days post-coitum.

 

Figure 2
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  		Fig. 2. ERK and ELK1 are phosphorylated during mouse preimplantation development. (A) Phosphorylated ERK (p-ERK, upper) and phosphorylated ELK1 (p-ElK-1, lower) were detected using anti-phospho-ERK antibody and anti-phospho-ELK1 antibody, respectively. Embryos (from left to right) at the two-cell, four-cell, eight-cell, morula or blastocyst stages were fixed and stained. Fluorescence was viewed with a confocal microscope. (B) Phosphorylation of ERK or ELK1 with or without U0126. Four-cell (top) and two-cell (bottom) stage embryos were treated with U0126 for 1 hour, and then the embryos were fixed and stained with either anti-phospho-ERK antibody (four cell) or anti-phospho-ELK1 antibody (two cell). Fluorescence was viewed with a confocal microscope.

 

Figure 3
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  		Fig. 3. ERK inactivation in late two-cell-stage embryos induces developmental arrest in the G2 phase, just before M phase in the four-cell stage. (A) BrdU incorporation into the nucleus was investigated. Late two-cell-stage embryos were cultured in the presence of BrdU and U0126 or BrdU and DMSO (control), fixed, and stained with anti-BrdU antibody. Fluorescence was viewed with a confocal microscope at the early and late four-cell stages. (B) Cyclin B1 (cyc B1) accumulation in the nucleus and phosphorylation of histone H3 (p-Histone H3) were examined. Late two-cell-stage embryos were cultured in the presence of U0126 or DMSO (control). Embryos were fixed and stained with anti-cyclin B1, Hoechst or anti-phospho-histone H3 antibody. Fluorescence was viewed with a confocal microscope (cyc B1) or a DeltaVision Image Restoration Microscope (Hoechst and p-Histone H3). Scale bars: 20 µm.

 

Figure 4
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  		Fig. 4. ERK inactivation in early two-cell-stage embryos induces developmental arrest in the G2 phase, just before M phase in the two-cell stage. (A) Early two-cell-stage embryos were treated with 20 µM U0126 or DMSO, and embryos were observed from E1.0 to E2.0. The zona pellucida was removed before inhibitor treatment. (B) BrdU incorporation into the nucleus was investigated. Early two-cell-stage embryos were cultured in the presence of BrdU and U0126 or BrdU and DMSO (control), fixed, and stained with anti-BrdU antibody. Fluorescence was viewed at the stages indicated with a confocal microscope. (C) Cyclin B1 accumulation in the nucleus and phosphorylation of histone H3 were examined. Early two-cell-stage embryos were cultured in the presence of U0126 or DMSO. Embryos were fixed and stained with anti-cyclin B1 or anti-phospho-histone H3 antibody. Fluorescence was viewed with a confocal microscope at the stages indicated.

 

Figure 5
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  		Fig. 5. The transcriptional program during mouse preimplantation development. (A) Actinomycin D completely blocked development. Two-cell-stage embryos were treated with actinomycin D (0.04 µM or 0.4 µM), and compared with control embryos (0 µM) at 24 (E2.5) and 48 (E3.5) hours after treatment. (B) Schedule of inhibitor treatment and of the microarray experiment. Solid and broken lines indicate the duration of U0126 and DMSO (vehicle) treatment, respectively. The inhibitor was added at the late two-cell stage, and embryos were collected for microarray experiments at the time points shown with red circles. (C) The numbers of genes whose expression levels in cont. 2.5 or cont. 3.5 were increased or decreased with statistical significance by more than threefold as compared with those in cont. 1.5 are shown. Because overlapping genes exist, in total, 6863 probe sets were differentially expressed. (D) Hierarchical clustering analysis showed the similarity in transcription profiles among the samples tested. This was performed by GeneSpring 7.3. dpc, days post-coitum; cont. 1.5/cont. 2.5/cont. 3.5, control embryos collected at day 1.5/2.5/3.5, respectively; U2.5, U0126-treated embryos collected at day 2.5; U3.5/U4.5, embryos released from the U0126-induced arrest, collected at day 3.5/4.5, respectively.

 

Figure 6
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  		Fig. 6. Genes whose expression profiles closely parallel the stages of embryonic development. (A) ERK-dependent genes were classified into eight groups by their differing expression patterns. Gray and yellow lines show patterns of control and U0126-treated gene expression profiles, respectively. (B, left) The expression pattern of each gene in each of the eight groups is displayed as a horizontal strip. Each of the treatment groups in represented vertically. For each gene, the ratio of mRNA level in the indicated sample to its level in control embryos at day 1.5 (cont. 1.5) is represented by a color, according to the color scale at the bottom. (Right) The bar graphs show the average expression profiles for the genes in the corresponding groups (1-8). cont. 1.5/cont. 2.5/cont. 3.5, control embryos collected at day 1.5/2.5/3.5, respectively; U2.5, U0126-treated embryos collected at day 2.5; U3.5/U4.5, embryos released from the U0126-induced arrest, collected at day 3.5/4.5, respectively.

 

Figure 7
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  		Fig. 7. Cell adhesion is important for early cell division cycles. (A) Genes that belong to the `cell junction' category in all of the eight groups of ERK-dependent genes were searched for by using GenMAPP/MAPPFinder software, and five genes were identified (red and purple). In the diagram, genes were color coded by the expression patterns that we assigned to each group. The figure is based on the Kyoto Encyclopedia of Genes and Genomes pathway database (http://www.genome.ad.jp/kegg/), with slight modification. (B) ECCD-1 treatment potentiates the sensitivity of embryos to U0126 treatment. Eight-cell-stage embryos were cultured for 24 hours in the presence of either 20 µM U0126 or ECCD-1 or both, and were then observed. The concentration of ECCD-1 used in the experiment was 1:500.

 

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© The Company of Biologists Ltd 2007