Xie Yufen, Wang Yingchun, Sun Tong, Wang Fangfei, Trostinskaia Anna, Puscheck Elizabeth, Rappolee Daniel A
CS Mott Center for Human Growth and Development of Ob/Gyn, Wayne State University School of Medicine, East Hancock, Detroit, Michigan 48201, USA.
Mol Reprod Dev. 2005 May;71(1):1-11. doi: 10.1002/mrd.20116.
Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in controlling embryonic proliferation and differentiation. It has been demonstrated that sequential lipophilic signal transduction mediators that participate in the MAPK pathway are null post-implantation lethal. It is not clear why the lethality of these null mutants arises after implantation and not before. One hypothesis is that the gene product of these post-implantation lethal null mutants are not present before implantation in normal embryos and do not have function until after implantation. To test this hypothesis, we selected a set of lipophilic genes mediating MAPK signal transduction pathways whose null mutants result in early peri-implantation or placental lethality. These included FRS2alpha, GAB1, GRB2, SOS1, Raf-B, and Raf1. Products of these selected genes were detected and their locations and functions indicated by indirect immunocytochemistry and Western blotting for proteins and RT-polymerase chain reaction (PCR) for mRNA transcription. We report here that all six signal mediators are detected at the protein level in preimplantation mouse embryo, placental trophoblasts, and in cultured trophoblast stem cells (TSC). Proteins are all detected in E3.5 embryos at a time when the first known mitogenic intercellular communication has been documented. mRNA transcripts of two post-implantation null mutant genes are expressed in mouse preimplantation embryos and unfertilized eggs. These mRNA transcripts were detected as maternal mRNA in unfertilized eggs that could delay the lethality of null mutants. All of the proteins were detected in the cytoplasm or in the cell membrane. This study of spatial and temporal expression revealed that all of these six null mutants post-implantation genes in MAPK pathway are expressed and, where tested, phosphorylated/activated proteins are detected in the blastocyst. Studies on RNA expression using RT-PCR suggest that maternal RNA could play an important role in delaying the presence of the lethal phenotype of null mutations.
丝裂原活化蛋白激酶（MAPK）信号通路在控制胚胎增殖和分化中起重要作用。已证明参与MAPK通路的一系列亲脂性信号转导介质在植入后是致死性缺失的。目前尚不清楚这些缺失突变体的致死性为何在植入后而非植入前出现。一种假说认为，这些植入后致死性缺失突变体的基因产物在正常胚胎植入前并不存在，直到植入后才具有功能。为了验证这一假说，我们选择了一组介导MAPK信号转导通路的亲脂性基因，其缺失突变体导致植入早期或胎盘致死。这些基因包括FRS2α、GAB1、GRB2、SOS1、Raf - B和Raf1。通过间接免疫细胞化学和蛋白质免疫印迹法检测这些选定基因的产物及其位置和功能，通过RT - 聚合酶链反应（PCR）检测mRNA转录。我们在此报告，在植入前的小鼠胚胎、胎盘滋养层细胞和培养的滋养层干细胞（TSC）中，所有六种信号介质均在蛋白质水平被检测到。在已知首次有丝分裂细胞间通讯被记录的E3.5胚胎中均检测到了这些蛋白质。两个植入后缺失突变基因的mRNA转录本在小鼠植入前胚胎和未受精卵中表达。这些mRNA转录本在未受精卵中作为母源mRNA被检测到，这可能会延迟缺失突变体的致死性。所有蛋白质均在细胞质或细胞膜中被检测到。这项关于时空表达的研究表明，MAPK通路中所有这六个植入后缺失突变基因均有表达，并且在囊胚中检测到了经测试的磷酸化/活化蛋白。使用RT - PCR对RNA表达的研究表明，母源RNA可能在延迟缺失突变致死表型的出现中起重要作用。
