Division of Cell Biology and Physiology, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Calcutta, West Bengal, 700032, India.
Academy of Scientific and Innovative Research (AcSIR), Sector 19, Kamla Nehru Nagar, Ghaziabad, UP, 201002, India.
Sci Rep. 2023 Jul 6;13(1):10978. doi: 10.1038/s41598-023-37977-2.
Trophectoderm cells of the blastocyst are the precursor of the placenta that is comprised of trophoblast, endothelial and smooth muscle cells. Since trophoectoderm cells are epithelial in nature, epithelial mesenchymal transition (EMT) of trophoblast stem (TS) cells might play pivotal role in placental morphogenesis. However, the molecular regulation of EMT during placental development and trophoblast differentiation still remained elusive. In this report, we sought to identify the molecular signature that regulates EMT during placental development and TS cell differentiation in mice. On E7.5 onwards the TS cells, located in the ectoplacental cone (EPC), rapidly divide and differentiate leading to formation of placenta proper. Using a real time PCR based array of functional EMT transcriptome with RNA from mouse implantation sites (IS) on E7.5 and E9.5, it was observed that there was an overall reduction of EMT gene expression in the IS as gestation progressed from E7.5 to E9.5 albeit the levels of EMT gene expression were substantial on both days. Further validation of array results using real time PCR and western blot analysis showed significant decrease in EMT-associated genes that included (a) transcription factors (Snai2, Zeb1, Stat3 and Foxc2), (b) extracellular matrix and cell adhesion related genes (Bmp1, Itga5, Vcan and Col3A1), (c) migration and motility- associated genes (Vim, Msn and FN1) and (d) differentiation and development related genes (Wnt5b, Jag1 and Cleaved Notch-1) on E9.5. To understand whether EMT is an ongoing process during placentation, the EMT-associated signatures genes, prevalent on E 7.5 and 9.5, were analysed on E12.5, E14.5 and E17.5 of mouse placenta. Interestingly, expression of these EMT-signature proteins were significantly higher at E12.5 though substantial expressions was observed in placenta with progression of gestation from mid- to late. To evaluate whether TS cells have the potential to undergo EMT ex vivo, TS cells were subjected to EMT induction, which was confirmed using morphological analysis and marker gene expression. Induction of EMT in TS cells showed similar gene expression profile of placental EMT. These results have broad biological implications, as inadequate mesenchymal transition leading to improper trophoblast-vasculogenic mimicry leads to placental pathophysiology and pregnancy failure.
囊胚的滋养外胚层细胞是胎盘的前体，由滋养层细胞、内皮细胞和平滑肌细胞组成。由于滋养外胚层细胞本质上是上皮细胞，因此滋养干细胞（TS）的上皮间质转化（EMT）可能在胎盘形态发生中发挥关键作用。然而，在胎盘发育和滋养细胞分化过程中 EMT 的分子调控仍然难以捉摸。在本报告中，我们试图确定调节胎盘发育和小鼠 TS 细胞分化过程中 EMT 的分子特征。在 E7.5 之后，位于胎盘外腔（EPC）的 TS 细胞迅速分裂和分化，导致胎盘的形成。使用基于实时 PCR 的功能 EMT 转录组阵列，对 E7.5 和 E9.5 时的小鼠着床部位（IS）的 RNA 进行分析，结果表明，随着妊娠从 E7.5 进展到 E9.5，IS 中的 EMT 基因表达总体上减少，尽管这两天的 EMT 基因表达水平都很高。使用实时 PCR 和 Western blot 分析对阵列结果进行进一步验证表明，包括（a）转录因子（Snai2、Zeb1、Stat3 和 Foxc2）、（b）细胞外基质和细胞黏附相关基因（Bmp1、Itga5、Vcan 和 Col3A1）、（c）迁移和运动相关基因（Vim、Msn 和 FN1）和（d）分化和发育相关基因（Wnt5b、Jag1 和 Cleaved Notch-1）在内的 EMT 相关基因显著减少。为了了解 EMT 是否是胎盘形成过程中的一个持续过程，分析了 E7.5 和 9.5 时存在的 EMT 相关特征基因在 E12.5、E14.5 和 E17.5 的小鼠胎盘上的表达情况。有趣的是，尽管在从中期到晚期的妊娠过程中胎盘中的表达量较高，但这些 EMT 特征蛋白的表达在 E12.5 时明显更高。为了评估 TS 细胞是否具有体外 EMT 的潜力，将 TS 细胞进行 EMT 诱导，通过形态分析和标记基因表达来验证。TS 细胞的 EMT 诱导显示出与胎盘 EMT 相似的基因表达谱。这些结果具有广泛的生物学意义，因为导致滋养细胞-血管生成模拟不当的间质转化不足会导致胎盘病理生理学和妊娠失败。
