G1期细胞周期蛋白对小鼠胚胎发育中第一次谱系分离的影响及机制研究

The first lineage segregation is a critical process to early mammalian development. It is related to the restricted expression of specific transcription factors, such as Cdx2. In previous studies, we found that after depletion of G1 cyclins, mouse embryonic stem cells showed high transcription levels of trophectoderm markers including Cdx2, and had the preference to differentiate into trophectoderm. This indicated that G1 cyclins might play a role in the first lineage segregation via regulating the expression of Cdx2. In this project, first, cyclin(s) with heterogeneity among blastomeres will be selected, and its correlation with Cdx2 in temporal and spatial expression pattern will be investigated. Then, the molecular mechanism of how G1 cyclins regulate Cdx2 will be explored, and CDX2 response elements to cyclins with will be identified. Lastly, to further confirm the involvement of G1 cyclins in first lineage segregation, ES cells and chimeric embryos with Cdx2 mutant lack of response elements to cyclins will be generated, and the phenotypes in pluripotency and differentiation will be analyzed. This project will provide evidence for the novel function of G1 cyclins in first cell fate decision, defining a new link between cell cycle and first lineage segregation in early development of mammals.

哺乳动物细胞的第一次谱系分离是早期胚胎发育的关键步骤，它的发生与特异性转录因子Cdx2等的限制性表达有关。在以往的研究中，我们发现小鼠胚胎干细胞（mESCs）在G1期细胞周期蛋白（G1 cyclins）全部缺失后，Cdx2转录水平明显上调，更倾向于向滋养外胚层分化，提示G1 cyclins可能通过调控Cdx2参与了谱系分离。本项目将首先筛选出从受精卵到囊胚过程中的卵裂球间表达异质性的G1期cyclin，观察其与Cdx2表达的时空相关性；然后在分子水平探索相关cyclin如何调控Cdx2，找出CDX2上的应答元件；最后，构建出应答元件缺失的CDX2突变体mESCs和嵌合胚胎，观察其表型，从体内体外方面，分析其分化发育和谱系分离能力，进一步验证G1 cyclins对Cdx2的调控机制，明确G1 cyclins对谱系分离的影响。这将对了解哺乳动物早期发育、再生和肿瘤等疾病都有重要意义。

在本项目中，我们通过分析各G1期细胞周期蛋白（G1 cyclins）（包括cyclin D1~3， E1~2）在从受精卵到囊胚发育的过程中，它们在每个卵裂球中的表达水平和分布情况，发现cyclin E1在小鼠早期胚胎发育过程中表达远高于其他G1 cyclins，并且随着cyclin E1表达的逐渐降低，Cdx2逐渐升高，在多种肿瘤细胞系中我们也发现了相同的表达模式，提示cyclin E1是抑制Cdx2表达和功能的主要G1期细胞周期蛋白；观察该G1 cyclin与Cdx2表达的时间空间相关性，通过蛋白质相互作用分析发现cyclin E1可能通过多种方式去抑制Cdx2的表达；构建该Cdx2应答元件缺失的突变体，在绿色荧光蛋白标记的小鼠胚胎干细胞基础上，获得小鼠胚胎模型，并再此基础上延展了在G1 cyclins 缺失细胞中敲除Cdx2探索其更多功能。此外，在培养小鼠胚胎干细胞过程中我们注意到细胞-细胞间接触和细胞外基质对维持小鼠胚胎干细胞的多能性具有着重要的作用。这将对了解哺乳动物早期发育、再生和肿瘤等的研究有重要科学价值。