Signaling-regulated establishment of pluripotency in vivo

项目总结 哺乳动物胚胎的首要任务是建立自己的胎盘，这一点最终将得到承认 并被母亲接受了。与此同时，胚胎必须通过赋予一些胎儿细胞来保留胎儿细胞 多能性。数十年的严谨研究揭示了部分解释这一现象的分子机制。 细胞命运的决定。在16-细胞期前后，胚胎还是一团细胞，细胞在外表面 极化和抑制河马信号。作为河马抑制的结果，转录因子YAP和 TAZ(WWTR1)进入外细胞核，与TEAD4配对，促进KEY的表达 胎盘(滋养外胚层)命运的调节者。令人兴奋的是，我们在老鼠身上发现的这种机制 在人类中保守，突出了小鼠模型作为发现平台的效用。然而，一个重大的谜团 尚未解决的问题：多能细胞是如何确定的？它们是不是因为不是多能的 滋养外胚层？还是有一种更积极的机制？我们的实验室发现多能性因子SOX2 在16-细胞期表现出独特的表达模式。也就是说，当其他多能性因素，如 Oct4和NANOG在胚胎的内外细胞中都有表达，SOX2仅在 在牢房里。这一观察结果表明，SOX2有助于积极确定多能性的最早阶段。 通过一系列实验，我们随后发现SOX2的表达模式依赖于 YAP/TAZ/TEAD转录复合体，表明滋养外胚层和多能性受 河马信号并行，从16细胞阶段开始。我们现在试图发现：1)SOX2模式 提供位置信息，以限制其他多能因子的活性 胚胎？2)SOX2类似物是否与SOX2或其他因子一起促进内细胞的多能性？ 3)YAP/TAZ/TEAD在促进SOX2表达的同时，如何机械地抑制Sox2的表达 外层细胞中的滋养外胚层基因？我们的团队汇集了高通量基因组编辑方面的专业知识， 单细胞基因表达的时间分辨分析，以及超低投入基因组学，这些都是 实现这里设想的主要机械性进步。这些研究将有助于揭示可概括的 哺乳动物胚胎发育的原则，是确保健康妊娠和儿童的基本原则 发展。

PROJECT SUMMARY The first priority of the mammalian embryo is to establish its own placenta, which will eventually be recognized and accepted by the mother. At the same time, the embryo must reserve fetal cells by endowing some with pluripotency. Decades of rigorous study have revealed the molecular mechanism that partly explains this first cell fate decision. Around the 16-cell stage, when the embryo is still a ball of cells, cells on the outer surface polarize and repress Hippo signaling. As a consequence of Hippo repression, transcription factors YAP and TAZ (WWTR1) enter the nuclei of the outer cells, where they partner with TEAD4 to promote expression of key regulators of placental (trophectoderm) fate. Excitingly, this mechanism, which we helped discover in mice, is conserved in humans, highlighting the utility of the mouse model as a discovery platform. Yet, a major mystery remained unsolved: how are the pluripotent cells specified? Are they pluripotent by virtue of being non- trophectoderm? Or is there a more active mechanism? Our lab discovered that the pluripotency factor SOX2 exhibits a unique expression pattern at the 16-cell stage. That is, when other pluripotency factors, such as OCT4 and NANOG are expressed in both inside and outside cells of the embryo, SOX2 is detected only in inside cells. This observation suggests that SOX2 helps actively specify the earliest stages of pluripotency. Through a series of experiments, we subsequently showed that SOX2 expression pattern is dependent on the YAP/TAZ/TEAD transcriptional complex, indicating that trophectoderm and pluripotency are regulated by Hippo signaling in parallel, starting at the 16-cell stage. We now seek to discover: 1) Does the SOX2 pattern provide the positional information to restrict the activity of other pluripotency factors to the inside cells of the embryo? 2) Do Sox2 paralogues work with SOX2 or other factors to promote the pluripotency of inside cells? 3) How do YAP/TAZ/TEAD mechanistically repress expression of Sox2 while promoting expression of trophectoderm genes in outer cells? Our team brings together expertise in high throughput genome editing, time-resolved analysis of single cell gene expression, and ultra-low input genomics, which are all needed to make the major mechanistic advances envisioned here. These studies will help reveal the generalizable principles of mammalian embryonic development that are fundamental to ensuring healthy pregnancy and child development.