极性蛋白PALS1通过Ras-MAPK信号通路调控小鼠胚胎ICM/TE世系分化的分子机制

Cell polarity is an essential property of mammalian cells. One of the most prominent features of cell polarity is the subdivision of cell membrane into apical and basolateral compartments by tight junctions. In mouse preimplantation development, cell polarity occurs from the 8-cell stage and after. Polarity proteins are involved in the asymmetric distribution of cytoplasmic determinants along apical-basolateral membrane and in the control of asymmetric divisions. Ultimately, cell polarity leads to ICM/TE lineage specification at the blastocyst stage. However, the exact molecular mechanism underlying ICM/TE lineage specification determined by cell polarity remains poorly investigated. The establishment of apical-basolateral polarity is dependent on the complex interplay of a number of molecules and macromolecular complexes. PALS1 is an essential member of CRB-PALS1-PATJ-AMOT-RICH1 apical polarity complex that localized to tight junctions. In the present project, we first examine the effect of knockout PALS1 on the development of mouse preimplantation embryos and ICM/TE lineage specification by quantitative PCR and embryo aggregation chimera. Then we investigate the regulation of PALS1 on polarity protein interaction and subcellular localization of polarity proteins to tight junctions and apical membrane in mouse embryos by using immunofluorescence and bimolecular fluorescence complementation (BiFC). Finally, we investigate the modulation of PALS1 on Ras-MAPK signaling pathway and whether this modulation signaling determines ICM/TE lineage fate in the mouse preimplantation embryo via influence on the expression and localization of lineage defining factors. The present project provides a systematic research plan on molecular mechanism underlying polarity protein PALS1 determination of ICM/TE lineage specification through Ras-MAPK signaling pathway in the mouse preimplantation embryo.

植入前胚胎发育中，细胞极性建立及其导致的ICM/TE世系分化事件对于动物的个体发育至关重要。胚胎细胞极性决定内外卵裂球向不同世系分化的信号途径和分子机制仍不明确。本项目以极性蛋白PALS1与Ras-MAPK信号通路的关系为研究切入点，拟以条件性基因敲除小鼠为模型，应用免疫细胞化学、定量PCR、胚胎嵌合体等技术研究PALS1对植入前胚胎细胞极性建立和世系分化的调控作用，检测PALS1对胚胎形态学发育及内在的极性蛋白复合体结构稳定性的影响，应用新颖的双分子荧光互补技术研究胚胎中极性蛋白的相互作用，并检测PALS1与极性蛋白互作对于Ras-MAPK信号通路的调节作用，研究这种调节作用对世系调控因子的表达定位及胚胎世系分化的影响，系统阐明极性蛋白PALS1通过Ras-MAPK信号通路决定植入前胚胎ICM/TE世系命运的分子机制。本研究在植入前胚胎细胞极性建立及世系命运决定的基础理论方面具有创新性。

胚胎细胞极性调控ICM/TE世系分化的信号传递途径和分子机制仍不明确。本项目探索了CRUMBS/PALS1/PATJ极性蛋白复合体对植入前胚胎细胞极性建立和世系分化的调控功能。研究结果主要包括：1）明确了复合体蛋白PALS1、CRB3、PATJ及其互作蛋白AMOT、RICH1等在植入前各期胚胎中的时序表达模式以及其沿着细胞顶端-基侧极性的不对称空间分布，证明了这些极性蛋白的表达定位严格受到发育调控；2）在此基础上，敲除或干扰极性蛋白复合体成员，检测胚胎极性是否出现错误及其形态学表型，筛选出阻滞ICM/TE世系分化的极性蛋白；3）采用双分子荧光互补技术检测了极性复合体成员RICH1和AMOT在致密化8细胞和桑椹胚的外部卵裂球的顶端膜相互作用，有趣的是，虽然在内部卵裂球的细胞膜上有RICH1和AMOT的分布，但二者之间不存在互作；4）揭示了Ras-MAPK通路的下游转录因子TFAP2c、Creb1、Sp3、ATF2在第一次世系分化中具有重要作用。