Role of VGLL1 in human placental development and trophoblast specification

项目摘要/摘要 许多与胎盘相关的妊娠并发症，包括先兆子痫(PE)和宫内 生长受限(IUGR)起源于胎盘发育的早期阶段，在 建立滋养层细胞，胎盘的功能构建块。由于 人类发展的这个早期阶段是不可接近的，我们所知道的关于这个过程的大部分 来自其他物种，特别是老鼠。植入前滋养层细胞的规格 小鼠胚胎依赖于Tead4/YAP1复合体启动滋养层细胞特异性 转录程序。然而，来自我们实验室和其他实验室的越来越多的证据表明， 小鼠和人之间滋养层细胞谱系规格的物种特异性显著差异。 在最近的一项胎盘形成比较研究中，我们确定了转录辅助因子残迹样 家族成员-1(VGLL1)作为人类早期妊娠细胞滋养层细胞(CTB)的特异性标志物 人类胎盘的干细胞室。VGLL1在早期与TEAD4和TP63共定位 妊娠CTB。此外，VGLL1的表达在滋养层细胞中很早就被诱导 骨形态发生蛋白4诱导前人多能干细胞的分化 TP63。人们对VGLL1功能知之甚少，但有趣的是，VGLL1已被证明与TEAD4结合， 以类似于滋养层细胞谱系所需的Tead4/YAP1相互作用的方式 在小鼠中的规格。该项目旨在阐明VGLL1作为人类特有的 滋养层细胞谱系规范的调节因子。我们将操作VGLL1在两个新- 建立人滋养层干细胞系，并在我们建立的基于hPSC的人滋养层干细胞模型中 滋养层细胞谱系规范。我们将评估这种基因操作对滋养层细胞的影响。 维持、分化和功能，以及全基因组RNA表达的变化。我们 将通过使用CRISPR/Cas9在人原代CTB中操纵VGLL1来验证我们的结果 技术我们将确认两个系统中VGLL1和TEAD4之间的交互，并确定 使用CHIP-SEQ直接靶向VGLL1-TEAD4复合体的下游。我们假设VGLL1 是参与人类早期发育中滋养层细胞谱系指定的关键角色，在 与TEAD4的复合体，以诱导基于TP63的转录程序建立和维持 人胎盘中的滋养层干细胞。

Project Summary/Abstract Many placenta-associated pregnancy complications, including pre-eclampsia (PE) and intrauterine growth restriction (IUGR), originate in the early stages of placental development, during the establishment of trophoblast cells, the functional building blocks of the placenta. Due to the inaccessibility of this early stage of human development, most of what we know about this process comes from other species, in particular, from mice. Trophoblast specification in pre-implantation mouse embryos is dependent upon the Tead4/Yap1 complex initiating a trophoblast-specific transcriptional program. However, an increasing body of evidence, from our lab and others’, shows significant species-specific differences in trophoblast lineage specification between mouse and human. In a recent comparative placentation study, we identified the transcriptional co-factor vestigial-like family member-1 (VGLL1) as a human-specific marker of early gestation cytotrophoblast (CTB), the stem cell compartment of the human placenta. VGLL1 co-localizes with TEAD4 and TP63 in early gestation CTB. Furthermore, expression of VGLL1 is induced very early during trophoblast differentiation of human pluripotent stem cells (hPSCs) following BMP4 treatment, before induction of TP63. Little is known about VGLL1 function, but interestingly, VGLL1 has been shown to bind TEAD4, in a manner similar to the Tead4/Yap1 interaction, which is required for trophoblast lineage specification in mice. This project aims to elucidate the role of VGLL1 as a human-specific regulator of trophoblast lineage specification. We will manipulate VGLL1 expression in both newly- established human trophoblast stem cell lines and in our established hPSC-based model of human trophoblast lineage specification. We will assess the effects of such gene manipulation on trophoblast maintenance, differentiation and function, as well as genome-wide changes in RNA expression. We will validate our results by manipulation of VGLL1 in human primary CTB using the CRISPR/Cas9 technology. We will confirm the interaction between VGLL1 and TEAD4 in both systems and identify direct downstream targets of the VGLL1-TEAD4 complex using ChIP-seq. We hypothesize that VGLL1 is a key player involved in trophoblast lineage specification in early human development, acting in complex with TEAD4, to induce a TP63-based transcriptional program to establish and maintain trophoblast stem cells in the human placenta.