Ananya Ghosh

Healthy progression of human pregnancy relies on cytotrophoblast (CTB) progenitor self-renewal and its differentiation toward multinucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, the underlying molecular mechanisms that fine-tune CTB self-renewal or direct its differentiation toward 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 the 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 births (gestational time ≤28 wk) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish the critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal versus differentiation. It plays positive instructive roles in promoting CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from attaining the STB fate.

Early pregnancy loss affects ∼15% of all implantation-confirmed human conceptions. However, evolutionarily conserved molecular mechanisms that regulate self-renewal of trophoblast progenitors and their association with early pregnancy loss are poorly understood. Here, we provide evidence that transcription factor TEAD4 ensures survival of postimplantation mouse and human embryos by controlling self-renewal and stemness of trophoblast progenitors within the placenta primordium. In an early postimplantation mouse embryo, TEAD4 is selectively expressed in trophoblast stem cell–like progenitor cells (TSPCs), and loss of Tead4 in postimplantation mouse TSPCs impairs their self-renewal, leading to embryonic lethality before embryonic day 9.0, a developmental stage equivalent to the first trimester of human gestation. Both TEAD4 and its cofactor, yes-associated protein 1 (YAP1), are specifically expressed in cytotrophoblast (CTB) progenitors of a first-trimester human placenta. We also show that a subset of unexplained recurrent pregnancy losses (idiopathic RPLs) is associated with impaired TEAD4 expression in CTB progenitors. Furthermore, by establishing idiopathic RPL patient-specific human trophoblast stem cells (RPL-TSCs), we show that loss of TEAD4 is associated with defective self-renewal in RPL-TSCs and rescue of TEAD4 expression restores their self-renewal ability. Unbiased genomics studies revealed that TEAD4 directly regulates expression of key cell cycle genes in both mouse and human TSCs and establishes a conserved transcriptional program. Our findings show that TEAD4, an effector of the Hippo signaling pathway, is essential for the establishment of pregnancy in a postimplantation mammalian embryo and indicate that impairment of the Hippo signaling pathway could be a molecular cause for early human pregnancy loss. Significance Reproductive success in placental mammals relies on proper development of the trophoblast lineage. In particular, a proper balance in self-renewal vs. differentiation of trophoblast progenitors is critical for establishment of pregnancy. A defect in this process causes early pregnancy loss. Here, we showed that the Hippo signaling effector, TEAD4, is essential for trophoblast progenitor self-renewal in early postimplantation mammalian embryos. Using genetic mouse models and human TSCs, including TSCs from patients with recurrent pregnancy losses, we identified a TEAD4-dependent, evolutionarily conserved gene expression program that promotes stemness and cell proliferation in trophoblast progenitors and ensures in utero survival of the developing fetus.

In utero mammalian development relies on the establishment of the maternal–fetal exchange interface, which ensures transportation of nutrients and gases between the mother and the fetus. This exchange interface is established via development of multinucleated syncytiotrophoblast cells (SynTs) during placentation. In mice, SynTs develop via differentiation of the trophoblast stem cell-like progenitor cells (TSPCs) of the placenta primordium, and in humans, SynTs are developed via differentiation of villous cytotrophoblast (CTB) progenitors. Despite the critical need in pregnancy progression, conserved signaling mechanisms that ensure SynT development are poorly understood. Herein, we show that atypical protein kinase C iota (PKCλ/ι) plays an essential role in establishing the SynT differentiation program in trophoblast progenitors. Loss of PKCλ/ι in the mouse TSPCs abrogates SynT development, leading to embryonic death at approximately embryonic day 9.0 (E9.0). We also show that PKCλ/ι-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. PKCλ/ι is selectively expressed in the first-trimester CTBs of a developing human placenta. Furthermore, loss of PKCλ/ι in CTB-derived human trophoblast stem cells (human TSCs) impairs their SynT differentiation potential both in vitro and after transplantation in immunocompromised mice. Our mechanistic analyses indicate that PKCλ/ι signaling maintains expression of GCM1, GATA2, and PPARγ, which are key transcription factors to instigate SynT differentiation programs in both mouse and human trophoblast progenitors. Our study uncovers a conserved molecular mechanism, in which PKCλ/ι signaling regulates establishment of the maternal–fetal exchange surface by promoting trophoblast progenitor-to-SynT transition during placentation.