Transcription profiling of mouse preimplantation development

Preimplantation development is a crucial step for successful implantation and pregnancy. Although both compaction and blastocyst formation have been extensively studied, mechanisms regulating early cell division stages before compaction have remained unclear. Here, we show that ERK MAP kinase function is required for early embryonic cell division and normal cell-cell adhesion before compaction. Our analysis demonstrates that inhibition of ERK activation in the late 2-cell stage embryos leads to a reversible arrest in G2 phase in the 4-cell stage. The G2 arrested, 4-cell stage embryos show weakened cell-cell adhesion as compared to control embryos. Remarkably, microarray analyses show that most of the programmed changes of upregulated and downregulated gene expression during the 4- to 8-cell stages normally proceed in the 4-cell stage-arrested embryos, except for a portion of the genes whose expression profiles closely parallel the stages of embryonic development when arrested in G2 and released to resume development. These parallel genes include the genes encoding intercellular adhesion molecules, whose expression is found to be positively regulated by the ERK pathway. We also show that while ERK inactivation in the 8-cell stage embryos does not lead to cell division arrest, it does cause cell division arrest when cadherin-mediated cell-cell adhesion is disrupted. These results demonstrate an essential role of ERK function in the G2/M transition and the expression of adhesion molecules during the 2-cell to 8-cell stage embryos, and suggest a loose parallelism between the gene expression programs and the developmental stages before compaction. Experiment Overall Design: We examined expression profiles of genes during early cell division stages in mouse preimplantation development. We performed the genome-wide analysis by using Affymetrix GeneChip oligonucleotide microarrays, and examined the effect of the ERK pathway inhibitor U0126 on the expression profiles. Two independent experiments were carried out. We collected embryos at six points as follows; control embryos at day 1.5 (cont. 1.5, 2-cell stage), day 2.5 (cont. 2.5, 4- to 8-cell), and day 3.5 (cont. 3.5, morula to blastocyst), and the U0126-treated embryos at day 2.5 (U2.5, 4-cell arrested), embryos released from the U0126-induced arrest at day 3.5 (U3.5, 8-cell) and at day 4.5 (U4.5, morula to blastocyst). Hybridization was carried out with the Mouse Genome 430 2.0 array following Affymetrix instructions. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Expression analysis was performed using GeneChip Operating Software v. 1.2 (GCOS) and GeneSpring 7.3.着床前胚胎发育（preimplantation development）是实现成功着床与妊娠的关键环节。尽管致密化（compaction）与囊胚（blastocyst）形成这两个过程已得到广泛研究，但调控致密化前早期细胞分裂阶段的分子机制仍未明确。本研究证实，ERK丝裂原活化蛋白激酶（ERK MAP kinase）的功能对于致密化前早期胚胎的细胞分裂及正常细胞间黏附不可或缺。分析结果显示，在晚期2细胞期胚胎中抑制ERK的激活，会使胚胎在4细胞期停滞于G2期，且该停滞状态可逆转。与对照组胚胎相比，G2期停滞的4细胞期胚胎的细胞间黏附能力显著减弱。值得注意的是，基因芯片（microarray）分析结果表明，4至8细胞期正常发生的上调与下调基因表达的程序性变化，在4细胞期停滞的胚胎中大多可正常进行；仅部分基因的表达谱与胚胎发育停滞于G2期并被释放以恢复发育时的阶段高度匹配。此类表达匹配的基因包括编码细胞间黏附分子的基因，经证实这类基因的表达受ERK通路正向调控。本研究同时发现，尽管在8细胞期胚胎中灭活ERK不会引发细胞分裂停滞，但当钙黏蛋白（cadherin）介导的细胞间黏附被破坏时，ERK灭活即可导致细胞分裂停滞。上述结果证实，ERK功能在2至8细胞期胚胎的G2/M转换及黏附分子表达中发挥核心作用，并提示致密化前胚胎的基因表达程序与发育阶段之间存在松散的相关性。 实验整体设计：我们针对小鼠着床前胚胎发育早期细胞分裂阶段的基因表达谱展开了研究。采用Affymetrix GeneChip寡核苷酸基因芯片开展全基因组表达分析，并探究了ERK通路抑制剂U0126对基因表达谱的影响。本研究共开展两组独立重复实验。我们按以下6个时间点收集胚胎：对照组胚胎分别采集于妊娠第1.5天（cont.1.5，2细胞期）、第2.5天（cont.2.5，4至8细胞期）及第3.5天（cont.3.5，桑椹胚至囊胚期）；U0126处理组胚胎分别采集于第2.5天（U2.5，4细胞停滞期）、从U0126诱导的停滞中释放后于第3.5天采集的胚胎（U3.5，8细胞期），以及第4.5天采集的胚胎（U4.5，桑椹胚至囊胚期）。我们按照Affymetrix官方操作指南，使用Mouse Genome 430 2.0芯片完成杂交反应。杂交完成后的芯片采用Affymetrix GeneChip扫描仪进行扫描。基因表达数据分析采用GeneChip Operating Software v.1.2（GCOS）及GeneSpring 7.3完成。