Gene expression in the placenta of WT and Ctnnb1 overexpression mice. Gene expression in the placenta of WT and Ctnnb1 overexpression mice

Wnt signaling are essential for the maintenance and differentiation of stem/progenitor cells, including trophoblast stem cells during placental development. Hyper-activation of Wnt signaling has been shown to relate with human trophoblast diseases. However, litter is known about the impact and underlying mechanisms of excessive Wnt signaling during placental trophoblast development. In the present work, we found that Sfrp1,5 double mutant mouse exhibited disturbed trophoblast differentiation in the placental ectoplacental cone (EPC), where the precursors of secondary trophoblast giant cells (TGCs) and trophoblast cells in the spongiotrophoblast layer are located. Employing mouse models expressing a trunked β-catenin with exon 3 deletion globally and trophoblast-specifically, combining cultured trophoblast stem cells, we found that hyper-activation of canonical Wnt pathway exhausted the trophoblast precursor cells in the EPC, resulting in the overabundance of giant cells at the expense of spongiotrophoblast cells. Further examination uncovered that hyper-activation of canonical Wnt pathway disturbed trophoblast differentiation in the EPC via repressing Mash2 expression. Collectively, our findings demonstrate that appropriate canonical Wnt-β-catenin pathway is essential for EPC trophoblast differentiation during placental development. Our work also has high clinical relevance, since abnormal Wnt signaling are often associated with trophoblast-related diseases. Overall design: To detect the gene profiles in WT and Ctnnb1 over expression placentas, placenta are collected and subjected to RNA-Seq. After aligned to mouse mm10 by HISAT2, RPKM value was calculated by Edger. Our results show that Ctnnb1 the key regulator for placentation by repressing Ascl2 expression. The expression of Ascl2 was further confirmed by qPCR and ChIP-qPCR. In lieu of Ctnnb1, there are also some other genes differentially expressed after Ctnnb1 overexpression, indicating the essential role of Ctnnb1. This RNA-Seq data provides fundamental information for our further physiological study of Ctnnb1. Total RNAs in whole placenta from two independent mouse were extracted by TRIzol and purified by ploy-A before subjected to RNA-Seq by HiSeq2500. The expression of level of each gene is normalized to RPKM.

Wnt信号通路（Wnt signaling）在胎盘发育过程中，对包括滋养层干细胞（trophoblast stem cells）在内的干/祖细胞的维持与分化发挥着至关重要的作用。已有研究证实，Wnt信号通路的过度激活与人类滋养层疾病存在关联。然而，目前对于过度激活的Wnt信号通路在胎盘滋养层发育过程中的影响及潜在分子机制，学界仍知之甚少。
本研究发现，Sfrp1、Sfrp5双突变小鼠的胎盘外胎盘锥（ectoplacental cone, EPC）内滋养层分化受到干扰，而该区域正是次级滋养层巨细胞（secondary trophoblast giant cells, TGCs）前体以及海绵滋养层内滋养层细胞的分布位点。
我们通过构建全身特异性及滋养层特异性表达外显子3缺失的截短型β-连环蛋白的小鼠模型，并结合体外培养的滋养层干细胞实验，证实经典Wnt通路的过度激活会耗竭EPC内的滋养层前体细胞，最终导致巨细胞数量过剩，而海绵滋养层细胞则以其为代价出现数量减少。
进一步研究揭示，经典Wnt通路的过度激活通过抑制Mash2的表达，干扰了EPC内的滋养层分化进程。
综上，本研究结果表明，适度的经典Wnt-β-连环蛋白通路对于胎盘发育过程中EPC内的滋养层分化不可或缺。本研究同时具备较高的临床转化价值，因为异常的Wnt信号通路常与滋养层相关疾病密切相关。
实验设计概述：为检测野生型（WT）与Ctnnb1过表达胎盘中的基因表达谱，我们收集胎盘样本并进行RNA测序（RNA-Seq）。将测序reads通过HISAT2比对至小鼠mm10参考基因组后，利用Edger软件计算RPKM值。
本研究结果显示，Ctnnb1通过抑制Ascl2的表达，成为胎盘形成的关键调控因子。Ascl2的表达水平进一步通过定量PCR（qPCR）及染色质免疫共沉淀定量PCR（ChIP-qPCR）得到验证。
除Ctnnb1外，Ctnnb1过表达后尚有其他一批差异表达基因，这进一步印证了Ctnnb1的核心调控作用。本RNA测序数据可为我们后续针对Ctnnb1的生理学研究提供基础数据支撑。
我们使用TRIzol试剂提取了两只独立实验小鼠的全胎盘总RNA，并通过poly-A富集进行纯化，随后利用HiSeq2500测序平台完成RNA测序。所有基因的表达水平均以RPKM值进行标准化。