The Transcription Factor NF-E2 Regulates Trophoblast Differentiation and Placental Vascularisation Independent of Platelets
Authors: Kashif Muhammed , Andrea Hellwig , Stefanie Herzog , Ilya Vinnikov , Khurrum Shahzad , Peter Nawroth , Isermann Berend 
Published: October 2019

Abstract
Embryonic growth restriction is associated with increased perinatal morbidity and mortality. We previously established that the embryonic lack of the transcription factor NF-E2 results in intrauterine growth restriction (IUGR) associated with a reduced placental vascularisation. The mechanism underlying the reduced placental vascularisation in embryos lacking NF-E2 remains unknown. The transcription factor NF-E2 is expressed in cells of the erythrocyte and megakaryocyte lineage. Absence of NF-E2 impairs thrombocytopoiesis secondary to a megakaryocyte defect, resulting in severe thrombocytopenia. The mechanisms underlying the IUGR and reduced placental vascularisation, in particular the role of thrombocytopenia, remains unknown.
Analysis of placental vascularisation and embryonic growth revealed a reduction of vascularisation in NF-E2−/− placenta as early as day 14.5 p.c., while the growth retardation of NF-E2−/− embryos was only observed at day 17.5 p.c. Therefore, the placental phenotype precedes embryonic growth retardation, consistent with a primary defect within the placenta. RT-PCR and in situ hybridization detected expression of NF-E2 in the labyrinthine layer and the junctional zone of the E 14.5 placenta as well as in murine trophoblast stem cells in vitro. To determine whether platelet deficiency or a trophoblast specific defect impairs placental vascularisation various in vivo experiments were conducted. Restoring platelet generation in NF-E2 knock out embryos using tetraploid aggregation or via in utero megakaryocyte transplantation does not rescue the vascularisation defect. Consistently, platelet depletion in NF-E2 expressing embryos is not sufficient to reduce placental vascularisation. Therefore, the impaired vascularisation in NF-E2 deficient placenta is independent of the platelet deficiency and results from NF-E2-deficieny in trophoblast cells.
Expression analysis (RT-PCR, in situ hybridisation) showed up regulation of syncytiotrophoblast marker Gcm1 and down regulation of labyrinthine layer marker Esx1 in NF-E2−/− placentae. Using electron microscopy we detected thickening of labyrinth trophoblast layers (syncytiotrophoblast layer II and III). To further delineate the mechanisms gene-expression analyses was performed. A number of genes known to be relevant for placental development were less expressed in the absence of NF-E2. Among these genes Fra-1, a member of the AP-1 dimeric transcription factor family with an established role for placental labyrinthine formation, was markedly reduced. Using EMSA we detected a marked reduction of AP-1 binding activity in placental extracts of NF-E2 deficient embryos. These data establish that NF-E2 and Fra-1 interact directly, as has been previously established, or indirectly, through NF-E2 dependent regulation of Fra-1 expression in trophoblast cells, to regulate placental vascularisation.
These experiments identify a role of the transcription factor NF-E2 in non-hematopoetic cells. NF-E2 regulates expression of genes relevant for placental development, including the transcription factor Fra-1. The absence of NF-E2 results in impaired placental vascularisation and enhanced syncytium formation independent of platelets through a trophoblast specific defect. These studies therefore identify a new NF-E2 dependent mechanism underlying IUGR.