Establishing the development basis for the morphological and functional asymmetry of the human chorion

摘要 项目3的目的是了解人类绒毛膜不对称的起源，以及 组成过程出现在怀孕早期。目的是检验光滑绒毛膜（SC） 或绒毛膜层）-胎儿膜的外表面-通过细胞滋养层（CTB）发育产生 与绒毛膜（VC）或胎盘的CTB不同的程序，导致解剖学 两个地区的差异。SC中的CTB形成复层上皮; VC中的CTB形成高度分支 绒毛膜绒毛（CV），其产生侵入子宫的绒毛外（EVT）亚群。的假设 最近的scRNA-sequencing支持了这一观点，该测序使得能够解剖细胞的类型和分子程序 在第二个三个月的居民SC与VC和他们的邻居。出乎意料的是，CTB祖细胞（CTB 1）的居民 在两个区室中有非常相似的转录组，但VC中的转录组分化为STB或EVT， SC中的那些分化为EVT和新的CTB 4群体。同样，驻留在SC中的EVT和 VC具有几乎相同的转录程序，但SC EVT没有侵入蜕膜，而是保留在蜕膜中。 与CTB 4细胞混合。初步数据表明CTB 4细胞主动抑制VC EVT迁移/侵袭。 为了检验主要假设，Aim 1将确定何时CTB 1细胞驻留在细胞中的不同轨迹。 SC vs. VC分歧。scRNA-seq、空间转录组学和免疫定位方法将被应用 来分析妊娠早期的绒毛膜囊通过将结果与我们的第二季度数据进行比较，我们将 了解绒毛膜绒毛的发育如何在SC中受到抑制，以及为什么CTB 1细胞在这个位置形成一个 复层上皮我们还将确定如何预先存在的炎症条件，子宫内膜异位症（与。 多囊卵巢综合征和肥胖症），影响CTB 1分化。目标2将剖析潜在的信号 SC与VC的CTB 1细胞的不同发育轨迹。这些实验将确定 通过使用RNA-seq和计算方法，该过程处于细胞自主或非自主控制下 为了揭示在CTB亚群之间和从邻近的蜕膜发出信号的潜在配体-受体对， 中胚层和免疫细胞。将在来自SC的CTB 1细胞上测试候选调节信号的作用。 或VC来源在滋养层干细胞和类器官模型中的作用。目标3将解剖 存在于SC与VC中的EVT的不同功能轨迹的潜在信号。初步证据 表明来自SC CTB的条件培养基抑制EVT侵袭。我们将使用 目的2沿着蛋白质谱分析方法来鉴定候选调节剂并测定其促进 或抑制EVT侵入。通过完成这个项目，我们将构建第一个人类基因组的分子图谱。 CTB分化，包括胎盘和胎膜。其重要意义在于， 确定与早孕丢失相关的这些调节机制中的缺陷和/或 异常炎症状态，如子宫内膜异位症，对生育能力和妊娠结局产生负面影响。

Abstract Project 3 aims to understand the origins of asymmetry in the human chorion and whether defects in the component processes arise early in pregnancy. The goal is to test the hypothesis that the smooth chorion (SC or chorion laeve)—the outer surface of the fetal membranes—arises via a cytotrophoblast (CTB) developmental program distinct from that of the CTBs of the villous chorion (VC) or placenta proper, resulting in the anatomical differences of the two regions. CTBs in the SC form a stratified epithelium; CTBs of the VC form highly branched chorionic villi (CV) that give rise to an extravillous (EVT) subpopulation that invades the uterus. The hypothesis is supported by recent scRNA-sequencing that enabled dissection of the types and molecular programs of cells resident in the 2nd trimester SC vs. VC and their neighbors. Unexpectedly, the CTB progenitors (CTB1) resident in both compartments have very similar transcriptomes, but those in the VC differentiate into STBs or EVTs while those in the SC differentiate into EVTs and a novel CTB4 population. Likewise, the EVTs resident in the SC and VC have nearly identical transcriptional programs, but instead of invading the decidua, the SC EVTs remain intermixed with CTB4 cells. Preliminary data suggest that CTB4 cells actively inhibit VC EVT migration/invasion. To test the main hypothesis, Aim 1 will determine when the distinct trajectories of the CTB1 cells resident in the SC vs. the VC diverge. scRNA-seq, spatial transcriptomics, and immunolocalization approaches will be applied to analyze the chorionic sac across early gestation. By comparing the results with our 2nd trimester data, we will learn how the development of chorionic villi is repressed in the SC and why CTB1 cells in this location form a stratified epithelium. We will also determine how a pre-existing inflammatory condition, endometriosis (vs. polycystic ovary syndrome and obesity), impacts CTB1 differentiation. Aim 2 will dissect the signals underlying the distinct developmental trajectories of CTB1 cells of the SC vs. the VC. The experiments will determine if this process is under cell autonomous or non-autonomous control by using RNA-seq and computational approaches to uncover potential ligand-receptor pairs that signal among CTB subpopulations and from neighboring decidual, mesodermal and immune cells. The effects of candidate regulatory signals will be tested on CTB1 cells from SC or VC sources in trophoblast stem cell and organoid models for their ability to skew fate. Aim 3 will dissect the signals underlying the distinct functional trajectories of EVTs resident in the SC vs. the VC. Preliminary evidence suggests that conditioned medium from SC CTBs inhibits EVT invasion. We will use the strategy described in Aim 2 along with protein profiling approaches to identify candidate regulators and assay their ability to promote or inhibit EVT invasion. By completing this project, we will have constructed the first molecular map of human CTB differentiation that includes the placenta and fetal membranes. The major significance lies in enabling identification of defects in these regulatory mechanisms that are associated with early pregnancy losses and/or abnormal inflammatory states such as endometriosis that negatively impact fertility and pregnancy outcomes.