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

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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.