The Transcription Factor OVOL2 Represses ID2 and Drives Differentiation of Trophoblast Stem Cells and Placental Development in Mice

Trophoblasts are the first cell type to be specified during embryogenesis, and they are essential for placental morphogenesis and function. Trophoblast stem (TS) cells are the progenitor cells for all trophoblast lineages; control of TS cell differentiation into distinct trophoblast subtypes is not well understood. Mice lacking the transcription factor OVO-like 2 (OVOL2) fail to produce a functioning placenta, and die around embryonic day 10.5, suggesting that OVOL2 may be critical for trophoblast development. Therefore, our objective was to determine the role of OVOL2 in mouse TS cell fate. We found that OVOL2 was highly expressed in mouse placenta and differentiating TS cells. Placentas and TS cells lacking OVOL2 showed poor trophoblast differentiation potential, including increased expression of stem-state associated genes (Eomes, Esrrb, Id2) and decreased levels of differentiation-associated transcripts (Gcm1, Tpbpa, Prl3b1, Syna). Ectopic OVOL2 expression in TS cells elicited precocious differentiation. OVOL2 bound proximate to the gene encoding inhibitor of differentiation 2 (ID2), a dominant negative helix-loop-helix protein, and directly repressed its activity. Overexpression of ID2 was sufficient to reinforce the TS cell stem state. Our findings reveal a critical role of OVOL2 as a regulator of TS cell differentiation and placental development, in-part by coordinating repression of ID2

Antiviral inflammation during early pregnancy reduces placental and fetal growth trajectories

Many viruses are detrimental to pregnancy and negatively affect fetal growth and development. What is not well understood is how virus-induced inflammation impacts fetal-placental growth and developmental trajectories, particularly when inflammation occurs in early pregnancy during nascent placental and embryo development. To address this issue, we simulated a systemic virus exposure in early pregnant rats (gestational day 8.5) by administering the viral dsRNA mimic polyinosinic:polycytidylic acid (PolyI:C). Maternal exposure to PolyI:C induced a potent antiviral response and hypoxia in the early pregnant uterus, containing the primordial placenta and embryo. Maternal PolyI:C exposure was associated with decreased expression of the maternally imprinted genes Mest, Sfrp2, and Dlk1, which encode proteins critical for placental growth. Exposure of pregnant dams to PolyI:C during early pregnancy reduced fetal growth trajectories throughout gestation, concomitant with smaller placentas, and altered placental structure at midgestation. No detectable changes in placental hemodynamics were observed, as determined by ultrasound biomicroscopy. An antiviral response was not evident in rat trophoblast stem (TS) cells following exposure to PolyI:C, or to certain PolyI:C-induced cytokines including IL-6. However, TS cells expressed high levels of type I IFNR subunits (Ifnar1 and Ifnar2) and responded to IFN-α by increasing expression of IFN-stimulated genes and decreasing expression of genes associated with the TS stem state, including Mest. IFN-α also impaired the differentiation capacity of TS cells. These results suggest that an antiviral inflammatory response in the conceptus during early pregnancy impacts TS cell developmental potential and causes latent placental development and reduced fetal growth

Antiviral inflammation during early pregnancy reduces placental and fetal growth trajectories

Many viruses are detrimental to pregnancy and negatively affect fetal growth and development. What is not well understood is how virus-induced inflammation impacts fetal-placental growth and developmental trajectories, particularly when inflammation occurs in early pregnancy during nascent placental and embryo development. To address this issue, we simulated a systemic virus exposure in early pregnant rats (gestational day 8.5) by administering the viral dsRNA mimic polyinosinic:polycytidylic acid (PolyI:C). Maternal exposure to PolyI:C induced a potent antiviral response and hypoxia in the early pregnant uterus, containing the primordial placenta and embryo. Maternal PolyI:C exposure was associated with decreased expression of the maternally imprinted genes Mest, Sfrp2, and Dlk1, which encode proteins critical for placental growth. Exposure of pregnant dams to PolyI:C during early pregnancy reduced fetal growth trajectories throughout gestation, concomitant with smaller placentas, and altered placental structure at midgestation. No detectable changes in placental hemodynamics were observed, as determined by ultrasound biomicroscopy. An antiviral response was not evident in rat trophoblast stem (TS) cells following exposure to PolyI:C, or to certain PolyI:C-induced cytokines including IL-6. However, TS cells expressed high levels of type I IFNR subunits (Ifnar1 and Ifnar2) and responded to IFN-α by increasing expression of IFN-stimulated genes and decreasing expression of genes associated with the TS stem state, including Mest. IFN-α also impaired the differentiation capacity of TS cells. These results suggest that an antiviral inflammatory response in the conceptus during early pregnancy impacts TS cell developmental potential and causes latent placental development and reduced fetal growth

Histone deacetylase 1 and 2 drive differentiation and fusion of progenitor cells in human placental trophoblasts

Cell fusion occurs when several cells combine to form a multinuclear aggregate (syncytium). In human placenta, a syncytialized trophoblast (syncytiotrophoblast) layer forms the primary interface between maternal and fetal tissue, facilitates nutrient and gas exchange, and produces hormones vital for pregnancy. Syncytiotrophoblast development occurs by differentiation of underlying progenitor cells called cytotrophoblasts, which then fuse into the syncytiotrophoblast layer. Differentiation is associated with chromatin remodeling and specific changes in gene expression mediated, at least in part, by histone acetylation. However, the epigenetic regulation of human cytotrophoblast differentiation and fusion is poorly understood. In this study, we found that human syncytiotrophoblast development was associated with deacetylation of multiple core histone residues. Chromatin immunoprecipitation sequencing revealed chromosomal regions that exhibit dynamic alterations in histone H3 acetylation during differentiation. These include regions containing genes classically associated with cytotrophoblast differentiation (TEAD4, TP63, OVOL1, CGB), as well as near genes with novel regulatory roles in trophoblast development and function, such as LHX4 and SYDE1. Prevention of histone deacetylation using both pharmacological and genetic approaches inhibited trophoblast fusion, supporting a critical role of this process for trophoblast differentiation. Finally, we identified the histone deacetylases (HDACs) HDAC1 and HDAC2 as the critical mediators driving cytotrophoblast differentiation. Collectively, these findings provide novel insights into the epigenetic mechanisms underlying trophoblast fusion during human placental development

Identifying Novel Transcriptional Regulators in Trophoblast Development

Proper development of the placenta is vital for pregnancy success. The placenta regulates exchange of nutrients and gases between maternal and fetal blood and produces factors necessary to sustain pregnancy. Thus, maldevelopment of the placenta is linked with serious obstetrical complications that can jeopardize the health of both mother and child. Additionally, the placenta develops in parallel with multiple embryonic organs, notably the heart. Improper placental development can leave the heart vulnerable to environmental insults and result in cardiovascular disease. Therefore, understanding how the placenta forms is of major health importance. The parenchymal cells of the placenta are called trophoblasts, which arise from trophoblast stem (TS) cells differentiating through one of two distinct lineage pathways: syncytiotrophoblast (ST), which regulates maternal-fetal nutrient transfer, and extravillous trophoblasts (EVTs), which remodel the uterine vasculature. However, the molecular mechanisms governing differentiation of TS cells are poorly understood. In this thesis, I show that glial cells missing-1 (GCM1) and OVO-Like 2 (OVOL2) are two transcription factors critical for morphogenesis of the human and mouse placenta, respectively. GCM1 regulates differentiation of human TS cells into both ST and EVTs. Particularly, GCM1 coordinates development and function of EVTs by regulating expression of the EVT regulator ASCL2 and the WNT antagonist NOTUM. In mice, OVOL2 is highly expressed within trophoblasts and loss of OVOL2 results in poor differentiation capacity and abrogated development of the placenta. OVOL2 mediates its effects on mouse placental morphogenesis, in-part, by regulating expression of the stem-associated factor Id2. Interestingly, Ovol2- deficient embryos die prematurely at embryonic day 10.5 due to major defects in heart formation, albeit Ovol2 expression is undetectable in the fetal heart. Poor development of the fetal heart is likely attributed to the absence of a placenta-specific microRNA, mmu-miR- 1249-3p, in extracellular vesicles isolated from Ovol2-deficient TS cells. I further show that placental-derived extracellular vesicles traffic to the embryo in vivo, where they may contribute to nascent heart formation. Collectively, my thesis provides insight into the mechanisms regulating TS cell biology and how the placenta and heart develop in concert to ensure proper development of the embryo