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First published online 16 March 2005
doi: 10.1242/dev.01729

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Requirement of the MAP kinase signaling pathways for mouse preimplantation development

Momoko Maekawa¹, Takuya Yamamoto¹, Takuji Tanoue^1,*, Yasuhito Yuasa², Osamu Chisaka¹ and Eisuke Nishida^1,

¹ Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
² Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

View larger version (54K): [in a new window]   Fig. 3. Gene transcription is essential for preimplantation development. (A) Eight-cell stage embryos were treated with actinomycin D (0.05 µg/ml or 0.5 µg/ml), and compared with control embryos (0 µg/ml) at morula stage (upper panel). In addition, morula stage embryos were treated with actinomycin D, and observed at blastocyst stage (lower panel). Low and high magnifications are shown. (B) Hierarchical clustering analysis. The transcripts used in this analysis were increased or decreased by SB203580, SP600125 or U0126 at least by twofold over baseline (wild type). (C) The transcripts were classified into six groups based on the sensitivity to the drugs as shown. The numbers of transcripts in each group in each experiment are shown (upper). The numbers of the transcripts that were classified into each group in at least two out of the three independent experiments are shown (lower).

View larger version (84K): [in a new window]   Fig. 1. SB203580 and SP600125 inhibit normal blastocoel formation. (A) Schedule of inhibitor treatment and analysis of preimplantation embryos. The program of developmental processes is shown at the top. Arrowheads indicate timing of compaction and that of cavitation. Bars in scheme 1, 2, 3 and 4 indicate the duration of the inhibitor treatment. At the end of the bars (indicated by arrowheads), embryos were observed and the typical images are shown in B. The zona pellucida was removed before the inhibitor was added to M16 medium. The inhibitor was added at the early eight-cell stage, and embryos were examined before the cavitation in scheme 1. In scheme 2, the inhibitor was added at the early eight-cell stage, and embryos were examined at the blastocyst stage. In scheme 3, the inhibitor was added at the pre-cavitation stage. In scheme 4, the inhibitor was added after initiation of cavitation. (B) Schematic representation of the phenotypes of embryos treated with each inhibitor. (C) Embryos treated with 20 µM SB203580 (+SB) or 25 µM SP600125 (+SP) were compared with control embryos (Cont.). 1, 2, 3 and 4 correspond to those in A. (D) Time-dependent observations of control, SB203580-treated or SP600125-treated embryos were performed. The inhibitor (SB or SP) was added at the eight-cell stage.

View larger version (45K): [in a new window]   Fig. 2. p38 and JNK are active during mouse preimplantation development. (A) The activated form of p38 was detected by using anti-phospho-specific p38 MAP kinase antibody. Embryos at the four-cell, eight-cell, morula or blastocyst stage were stained with anti-phospho-specific p38 MAP kinase antibody. Fluorescence was viewed with a confocal microscope. (B) Phosphorylation of MAPKAPK-2 or HSP27 with or without SB203580. The eight-cell stage embryos were treated with SB203580 for 1.5 hours, and then the embryos were fixed and stained with anti-phospho-specific MAPKAPK-2 (Thr 334) antibody or anti-phospho-specific HSP27 (Ser 82) antibody. (C) Activation of JNK was assessed by using anti-phospho-specific Jun (Ser 73) antibody. Embryos at the four-cell, eight-cell, morula and blastocyst stage were stained with anti-phospho-specific Jun (Ser 73) antibody. Fluorescence was viewed with a confocal microscope. (D) Phosphorylation of Jun was examined with or without SP600125. SP600125 was added at the eight-cell stage. After 2 hours of incubation, the embryo was fixed and stained with anti-phospho-specific Jun (Ser 73) antibody. (E) Effect of JNK1/2-targeting siRNAs on mouse preimplantation development. One-cell stage embryos were not injected or injected with DDW or dsRNA oligonucleotides as indicated, and cultured for 3 days. The numbers of morphologically normal or abnormal embryos were counted. Bright-field microscopic photographs are shown.

View larger version (50K): [in a new window]   Fig. 4. Identified genes possibly function in cavity formation. The graphs are representative of the average expression profiles for the genes from each group (left). Similar results were obtained in two other experiments. A list of transcripts in each group (1-6) is shown (right). Detailed data are shown in Table S1 in the supplementary material.

View larger version (29K): [in a new window]   Fig. 5. Functional analysis of several genes in group 4. (A) Eight-cell stage embryos were treated with or without SB203580 or SP600125 for 1 hour, and stained with anti-CDX1 antibody or anti-Dkk1 antibody. Fluorescence was viewed with a confocal microscope. (B) Effect of siRNA-based inhibition of genes in group 4. One-cell stage embryos were injected with dsRNA oligonucleotides as indicated, and cultured for 3 days. The numbers of morphologically normal or abnormal embryos were counted, and embryos were stained with anti-Dkk1 antibody. Bright-field (upper) and fluorescence (lower) micrographs of DDW-injected (left) and siCdx1/siDkk1/siFoxq1/siSox7-injected (middle and right) embryos are shown.