Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku, Kita 9, Nishi 9, Sapporo, 060-8589, Japan.
Biochem Biophys Res Commun. 2020 Aug 6;528(4):713-718. doi: 10.1016/j.bbrc.2020.05.210. Epub 2020 Jun 6.
Mammalian blastocysts are composed of two distinct cell lineages, namely the inner cell mass (ICM) and trophectoderm (TE). TE cells that give rise to the embryonic placenta are marked by an exclusive expression of the key determinant transcription factor, CDX2. Although Hippo signaling pathway is known to be responsible for this TE-specific expression of CDX2, the upstream regulator of this pathway in mammalian embryos is still controversial. In the present study, the involvement of the small molecular G protein, RHOA, in TE cell-fate decision in cattle was investigated. Inhibition of RHOA by the specific inhibitor, C3 transferase (C3), severely impaired the blastocyst formation. Further, C3 treatment significantly decreased the number of blastomeres with nuclearized YAP1, the prominent effector of Hippo pathway. An artificial isolation of ICM cells from blastocysts followed by the continuing culture to regenerate TE cells was conducted and showed that TE re-emergence from the isolated ICM is governed by Hippo pathway and suppressed by C3 treatment like that observed in developing embryos. Finally, the long-term exposure to C3 suggests the presence of alternative regulators of CDX2 expression other than RHOA signaling because there were still CDX2-positive cells after C3 treatment. These results demonstrated that RHOA signaling plays a significant role in TE cell-fate decision by regulating Hippo pathway in cattle.
哺乳动物囊胚由两个不同的细胞谱系组成，即内细胞团（ICM）和滋养外胚层（TE）。产生胚胎胎盘的 TE 细胞通过关键决定转录因子 CDX2 的独特表达来标记。尽管 Hippo 信号通路负责 CDX2 在 TE 中的这种特异性表达，但哺乳动物胚胎中该通路的上游调节剂仍存在争议。在本研究中，研究了小分子 G 蛋白 RHOA 在牛 TE 细胞命运决定中的作用。特异性抑制剂 C3 转移酶（C3）抑制 RHOA 严重损害了囊胚的形成。此外，C3 处理显著减少了核化 YAP1 的卵裂球数量，YAP1 是 Hippo 通路的主要效应物。从囊胚中人工分离 ICM 细胞并继续培养以再生 TE 细胞的实验表明，TE 从分离的 ICM 中重新出现受 Hippo 通路调控，并像在发育中的胚胎中一样受到 C3 处理的抑制。最后，长期暴露于 C3 表明存在除 RHOA 信号以外的 CDX2 表达的替代调节剂，因为在 C3 处理后仍然存在 CDX2 阳性细胞。这些结果表明，RHOA 信号通过调节 Hippo 通路在牛中在 TE 细胞命运决定中发挥重要作用。
