转录因子ZEB2介导的调控猪胎盘褶皱绒毛膜上皮细胞形状转换的机制研究

The development of the placental folded structure is of importance in pigs to expand the maternal-fetal exchange surface area and then to maintain the pregnancy and fetal survival.The shape of chorionic epithelial cell changes during the development of placenta and have the impact on the formation of the placental folded structure.Our previous work found that in Large White pigs of day 95 gestation , the shape of chorionic epithelial cells at the top of the folded placenta was columnar but was cuboidal at the bottom of the folded placenta. However, in Meishan pig, all of the chorionic epithelial cells showed cuboidal shape.The proteins for cell adhesion such as E-cadherin were highly expressed in columnar chorionic epithelial cells, while the E-cadherin transcriptional repressor of ZEB2 was found to be highly expressed in cuboidal chorionic epithelial cells, indicating that ZEB2 may induce cell shape change by regulation of the expression of the proteins for cell adhesion. Therefore, the study will investigate the signaling pathway mediated by ZEB2 in regulation of chorionic epithelial cell shape change by using the ChIP-Seq, RNA-Seq and EMSA approaches .Characterization of the mechanism involved will carried out in the cell culture model, the pig placental tissue, and conditional gene knockout animal model. Knowledge gained will be of significance for the characterization of the mechanism regulation of the placental development and for the improvement of sow reproductive efficiency.

胎盘皱褶的形成是猪胎盘发育的重要特征，对胎儿发育与存活起关键作用，胎盘绒毛膜上皮细胞形状由柱状向立方状转换是皱褶形成的结构基础。申请人前期研究发现妊娠后期的绒毛膜上皮细胞形状在大白猪胎盘皱褶顶端为柱状、底侧部为立方状，而在梅山猪中则全部为立方状，胞间连接蛋白如E-cadherin等只在柱状绒毛膜上皮细胞中高表达，而其负调控转录因子ZEB2却只在立方状细胞中高表达，表明ZEB2可能靶向胞间连接基因，调控绒毛膜上皮细胞形状的改变。本项目拟以梅山和大白猪妊娠后期的胎盘绒毛膜为材料，采用荧光激活细胞分选技术获得两种形状的绒毛膜上皮细胞，结合RNA-Seq和ChIP-Seq及EMSA等技术分析ZEB2的靶基因及上游调控因子，在猪细胞和组织学水平及条件性基因敲除动物模型中鉴定ZEB2介导的调控猪胎盘褶皱绒毛膜上皮细胞形状转换的信号通路，为解析猪胎盘发育的机制奠定基础，为母猪繁殖效率的遗传改良提供依据。

猪胎盘发育的重要特征是胎盘皱褶的形成，本项目对相关机制展开研究。（1）获得了猪妊娠期母胎界面组织细胞学特征数据，发现绒毛膜上皮细胞在品种及妊娠期间的形状及分泌能力存在差异。（2）发现猪ZEB2的5’UTR中存在抑制ZEB2翻译的调控元件，而lncRNA ZEB2-AS可以影响ZEB2的表达。利用免疫共沉淀（ChIP）技术及EMSA等技术证实在猪胎盘中表达的转录因子ZEB2可以通过靶向E-cadherin启动子中的E2-box区域调控E-cadherin/β-catenin复合物的表达从而在绒毛膜上皮细胞形状改变中发挥作用。（3）利用RNA-seq和ChIP-seq技术获得了梅山猪胎盘褶皱形成期（绝大部分绒毛膜上皮细胞为柱状）和扩展期（绝大部分绒毛膜上皮细胞为立方状）胎盘绒毛膜转录组及全基因组水平组蛋白H3K4me3和H3K27ac修饰图谱，检测到时期间组蛋白差异修饰的基因组区域主要富集在血管渗透性、细胞形态和细胞粘附相关通路的基因和lncRNA中，表明H3K4me3和H3K27ac在调控猪胎盘发育和功能中具有重要作用。（4）全基因组关联分析（GWAS）检测到与猪初生重这一胎盘发育重要指标显著关联的基因组区域，整合绒毛膜RNA-seq和ChIP-seq数据后鉴定到与胎盘发育关联的基因模块和突变。（5）发现FUT8和转录因子samd2的磷酸化水平与猪胎盘绒毛膜上皮细胞的形态变化相关，可能影响基质金属蛋白酶13的表达及IV型胶原和层粘连蛋白在上皮细胞基底膜中的表达。CDC42基因是Rho家族中鸟嘌呤三核苷酸激酶，HAI-1是一种Kunitz 型丝氨酸蛋白酶抑制因子，都具有调控细胞迁移或形态的作用。我们发现这两个基因在猪胎盘立方状和柱状绒毛膜上皮细胞中差异表达， CDC42基因3’UTR的miR-18a的结合区域种子序列中的一个SNP 位点导致miR-18a与CDC42的互作效应存在多态性且与猪产仔数显著关联，而HAI-1基因3’UTR区的1个SNP与窝平均初生重显著关联。本项目发现了包括ZEB2在内的对猪胎盘发育有重要作用的基因和信号通路，可为解析猪胎盘发育的机制提供依据，为猪繁殖性状的遗传改良提供了新的标记。