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

摘要 项目3旨在了解人类绒毛膜不对称的起源，以及 组成过程在怀孕早期就开始了。我们的目标是测试平滑绒毛膜(SC)的假设 或绒毛膜)--胎儿膜的外表面--通过细胞滋养层(CTB)发育而产生 程序不同于绒毛绒毛(VC)或胎盘本身的CTB，导致解剖学上的 这两个地区的差异。SC中的CTB形成复层上皮；VC中的CTB形成高度分枝 绒毛膜绒毛(CV)形成侵入子宫的绒毛外(EVT)亚群。假说 得到了最近的scRNA测序的支持，这使得对细胞的类型和分子程序的剖析成为可能 居住在怀孕中期的SC VS VC和他们的邻居。出乎意料的是，CTB前身(CTB1)常驻 在两个隔室中有非常相似的转录本，但在VC中的转录本分化为STB或EVT，而 SC中的基因分化为EVT和新的CTB4群体。同样，驻留在SC和 VC有几乎相同的转录程序，但SC EVT没有侵入蜕膜，而是保留了 与CTB4细胞混合。初步数据表明，CTB4细胞能有效抑制VC-EVT的迁移/侵袭。 为了检验主要假设，目标1将确定CTB1细胞的不同轨迹何时驻留在 供应链与风投之间存在分歧。将应用scrna-seq、空间转录学和免疫定位方法。 来分析妊娠早期的绒毛膜囊。通过将结果与我们的中期妊娠数据进行比较，我们将 了解绒毛的发育是如何在SC中被抑制的，以及为什么这个位置的CTB1细胞形成一个 复层上皮。我们还将确定先前存在的炎症性疾病--子宫内膜异位症(vs. 多囊卵巢综合征和肥胖)，影响CTB1的分化。目标2将剖析潜在的信号 SC和VC的CTB1细胞的不同发育轨迹。实验将确定这是否 通过使用rna-seq和计算方法，过程处于细胞自主或非自主控制之下。 为了发现CTB亚群和邻近蜕膜中潜在的配体-受体对发出信号， 中胚层和免疫细胞。候选调控信号的影响将在来自SC的CTB1细胞上进行测试 或VC来源滋养层干细胞和有机体模型，因为它们有能力扭转命运。《目标3》将剖析 驻留在SC和VC中的EVT的不同功能轨迹背后的信号。初步证据 提示SC CTB的条件培养液可抑制EVT的侵袭。我们将使用中介绍的策略 目标2和蛋白质谱方法，以确定候选调节子并分析它们促进 或抑制EVT的侵袭。通过完成这个项目，我们将构建第一个人类分子图谱 CTB分化包括胎盘和胎膜。其主要意义在于使 确定这些调节机制中与早孕丢失和/或 异常炎症状态，如子宫内膜异位症，对生育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.