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

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.

Grant Number: 3P50HD112034-02S1
NIH Institute/Center: NIH
Principal Investigator: Robert Blelloch