Published On: Tue, Apr 16th, 2019

Dominant-negative inhibition of canonical Notch signaling in trophoblast cells does not disrupt placenta formation [RESEARCH ARTICLE]


The development and function of the placenta relies heavily on embryo-derived trophoblasts (TBs), specialized cells that interact with maternal endothelial cells (ECs) to form mosaic vessels that facilitate proper blood flow to a developing conceptus (Cha et al., 2012; Cross et al., 1994; Hunkapiller and Fisher, 2008; Rai and Cross, 2014). In humans and mice, invasive TB subtypes mediate uterine vascular remodeling. Reduced TB-mediated uterine vascular invasion leads to poor placental vascular development and improper placentation, which are associated with miscarriages, intrauterine growth restriction and pre-eclampsia (Cha et al., 2012; Cross et al., 1994; Rossant and Cross, 2001). The signaling pathways active in the EC-TB crosstalk that underlies placentation have not been fully elucidated. However, Notch signaling, a regulator of angiogenesis and vascular remodeling, has been implicated in both human and mouse placenta formation (Chi et al., 2017; Gasperowicz and Otto, 2008; Gasperowicz et al., 2013; Haider et al., 2014, 2017; Hunkapiller et al., 2011; Kalkunte et al., 2017; Lee et al., 2018).

The Notch pathway regulates cell fate, cellular growth and invasion via direct cell-to-cell contact (Cuman et al., 2014; Kofler et al., 2011). Notch proteins (Notch1-4) are single-pass transmembrane receptors that are activated by membrane bound ligands of the Jagged and Delta-like ligand (Dll) families on adjacent cells (Shawber and Kitajewski, 2004). With canonical Notch signaling, binding of the ligand to Notch triggers cleavage and translocation of the Notch intracellular domain (NICD) to the nucleus, where it binds to the transcription factor recombination binding protein jκ (Rbpjκ). NICD binding and recruitment of co-activators, including Mastermind-like1 (MAML1), converts Rbpjκ from a transcriptional repressor to an activator, inducing downstream targets of the Hairy/Enhancer of Split (Hes) and Hairy/Enhancer of Split related with YRPW motif (Hey) families (Kofler et al., 2011; Kovall, 2007). In addition to Rbpjκ-mediated canonical Notch signaling, both the full-length Notch proteins and the NICD can signal via a non-canonical Rbpjk-independent pathway to regulate cell fate (Andersen et al., 2012; Shawber et al., 1996).

In the mouse, Notch signaling is active in both ECs and TBs (Hunkapiller et al., 2011; Levin et al., 2017) and Notch function is critical for development of the placental vascular system and proper placenta formation (Chi et al., 2017; Gasperowicz and Otto, 2008; Haider et al., 2017; Hunkapiller et al., 2011). Notch expression and activity have been detected in TBs in the ectoplacental cone, TBs associated with maternal decidual vessels and TBs in the junctional zone (Hunkapiller et al., 2011; Levin et al., 2017) (Fig. 1A). Global deletion of Notch1, Notch1/Notch4, Dll4, or Rbpjκ resulted in embryonic lethality by mid-gestation via disruption in chorioallantoic branching and/or placental labyrinth network formation (Duarte et al., 2004; Gasperowicz and Otto, 2008; Krebs et al., 2000; Limbourg et al., 2005; Oka et al., 1995). Placentas lacking Tle3, a transcriptional co-repressor that interacts with downstream effectors of the Notch signaling pathway, have reduced junctional zone size and abnormal TB-lined maternal vasculature (Gasperowicz et al., 2013). Notch2 null embryos died by embryonic day (E) 11.5 and whole embryo culture rescued lethality in mutant embryos, suggesting an extraembryonic cause (Hamada et al., 2007). Conditional deletion of Notch2 with the TB-specific Tpbpa-Cre driver resulted in reduced size of maternal vessels and decreased placental perfusion, suggesting a requirement for Notch2 in differentiation of invasive TB subtypes and TB-mediated remodeling of the maternal vasculature (Hunkapiller et al., 2011).

Herein, we used Cyp19-Cre and Tpbpa-Cre, two TB-specific Cre drivers with overlapping yet unique TB expression patterns, to investigate the requirement for TB-specific canonical Notch signaling in placentation and placental function. Cyp19-Cre induces recombination in many TB stem cell derivatives, including spongiotrophoblasts, labyrinthine TBs and many trophoblast giant cells (TGCs), including parietal-TGCs and spiral artery associated-TGCs (Moreau et al., 2014; Wenzel and Leone, 2007). Tpbpa-Cre expression includes invasive TB subtypes, glycogen trophoblast cells and spiral artery associated-TGCs, found in the mature placenta (Hu and Cross, 2010; Hunkapiller et al., 2011; Simmons et al., 2007). We hypothesized that placentation requires Notch in TBs and that inhibition of TB-specific canonical Notch signaling would severely impact placental development and pregnancy outcomes. In this study, we report the use of a dominant-negative form of MAML1 (DNMAML) to inhibit TB-specific canonical Notch signaling using both Cyp19-Cre and Tpbpa-Cre drivers. As the Cyp19-Cre transgene has broader expression, we predicted that the phenotype resulting from disrupted canonical Notch signaling could be more severe with the Cyp19-Cre driver than with the Tpbpa-Cre driver.

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