OCT4在猪滋养层发育中的功能及作用网络

The exclusively expression of Oct4 and Cdx2 in mouse embryonic cells is crucial for inner cell mass （ICM） and trophectoderm（TE ）differentiation and results in Oct4 specially expressed in ICM and Cdx2 specially expressed in TE. But OCT4 keep expressing in TE in large mamalian embryos represented by pig. According to this biological phenomenon, we proposed the scientific question: what is the biological importance of OCT4 keep expressing in porcine TE cells and what is the molecular net works that OCT4 functioned through in TE. Based on the scientific question, in this study the accurate expression pattern of OCT4 in TE of porcine blastocyst will be examined; the funtion of OCT4 on embryonic development will be analyzed by the development results of TE specific OCT4 over expression or knock down embryos. The function of OCT4 on TE cells will be explored by the potential of TE chimeric development and the characteristics of TE derived stem cell lines. The molecular net works of OCT4 functioned in TE will be investicated by RNA-Seq and ChIP-Seq. OCT4 down stream candidate genes will be screened and verificated by routine way and finally OCT4 regulation net works will be constructed.

OCT4和CDX2两种转录因子在小鼠早期胚胎细胞中互斥性表达对于内细胞团（ICM）和滋养层（TE）分化具有重要作用，在囊胚中OCT4特异性表达于ICM，而CDX2特异性于TE表达。但在以猪为代表的其它大型哺乳动物，OCT4持续在囊胚TE中表达。据此，我们提出如下科学问题：OCT4在猪囊TE中持续表达的生物学意义和发挥功能的分子网络途径。围绕该科学问题，本研究将确认OCT4在猪囊胚TE中的精确表达模式，通过特异性调控OCT4在TE中的表达，跟踪胚胎发育表型变化，推断其对胚胎发育的生物学作用；通过TE细胞嵌合体制作和TE细胞体外培养建系探索OCT4对TE细胞的生物学作用；通过RNA-Seq测序和ChIP-Seq建立OCT4在TE细胞中发挥生物学功能的分子调控网络；筛选并验证OCT4下游候选调控基因作用关系，最终初步建立猪TE细胞中OCT4发挥功能的分子调控网络。

OCT4在小鼠囊胚中呈现内细胞团特异性表达模式，而在猪囊胚中则呈现滋养层和内细胞团同时表达模式。为了探索OCT4在猪囊滋养层中的生物学功能和分子机制，本项目首先通过多种技术路线跟踪了OCT4在猪囊胚滋养层中的表达模式，然后围绕OCT4对滋养层发育潜能的影响和对胚胎整体发育的作用展开了系统研究，通过对滋养层特异性敲低OCT4胚胎的高通量测序分析探索了OCT4在滋养层中发挥生物学功能的分子机制。研究结果表明OCT4在扩展囊胚期之后仍呈现高量表达，特异性敲低滋养层中OCT4的表达会导致囊胚发育阻滞，而在滋养层中特异性过表达OCT4可以提升囊胚质量；分离囊胚滋养层细胞并注射到桑椹胚中，滋养层细胞可以参与到嵌合体胚胎的内细胞团发育，表明其具有发育的可塑性；在LCDM培养体系中培养猪早期囊胚可以获得多潜能干细胞系，该干细胞系具有滋养层分化倾向；对滋养层特异性敲低OCT4胚胎进行RNA-Seq分析，结果发现了12个猪滋养层中特异性表达的差异基因，其中hnRNPA2B1被初步证明是OCT4在滋养层中发挥生物学功能的重要下游基因。上述研究结果深化了OCT4调控猪滋养层发育机制的认知，对于在畜牧生产中研发提高猪胚胎着床率的技术具有现实意义。