Hippo Signaling Cofactor, WWTR1, at the Crossroads of Human Trophoblast Progenitor Self-Renewal and Differentiation

ABSTRACT Healthy progression of human pregnancy relies on cytotrophoblast progenitor (CTB) self-renewal and their differentiation towards multi-nucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, underlying molecular mechanisms that fine-tune CTB self-renewal or direct their differentiation towards STBs or EVTs during human placentation are poorly defined. Here, we show that hippo signaling cofactor WW Domain Containing Transcription Regulator 1 (WWTR1) is a master regulator of trophoblast fate choice during human placentation. Using human trophoblast stem cells (human TSCs), primary CTBs and human placental explants, we demonstrate that WWTR1 promotes self-renewal in human CTBs and is essential for their differentiation to EVTs. In contrast, WWTR1 prevents induction of STB fate in undifferentiated CTBs. Our single-cell RNA-sequencing analyses in first-trimester human placenta along with mechanistic analyses in human TSCs revealed that WWTR1 fine-tunes trophoblast fate by directly regulating Wnt signaling components. Importantly, our analyses of placentae from pathological pregnancies show that extreme preterm birth (gestational time ≤28weeks) and intrauterine growth restriction along with preeclampsia (IUGR/PE) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish a critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal vs. differentiation. It plays positive instructive roles to promote CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from obtaining the STB fate. SIGNIFICANCE Human pregnancy relies on formation of the transient organ placenta and trophoblast cells are the major building blocks of the placenta. A defect in trophoblast progenitor self-renewal or their differentiation is associated with either pregnancy loss or pathological pregnancies, yet underlying molecular mechanisms that regulate trophoblast differentiation are poorly understood. In this study, we discovered that WWTR1, a transcription cofactor and a component of conserved Hippo signaling pathway, optimizes trophoblast progenitor self-renewal and is essential for their differentiation into the invasive extravillous trophoblast cell lineage. Our findings establish WWTR1 as a critical regulator for success in human placentation and progression of a healthy pregnancy.