AKT1-FOXO4 Axis Regulates Hemochorial Placentation [Foxo4-KD-rTSC]

AKT1 is a serine/threonine kinase implicated in fetal, placental, and postnatal growth. In this study, we investigated roles for AKT1 in placental development using a genome-edited/loss-of-function rat model. Both heterozygous and homozygous Akt1 mutant rats were viable and fertile. Disruption of AKT1 resulted in placental, fetal, and postnatal growth restriction. Akt1 null placentas showed deficits in both junctional zone and labyrinth zone size and their ability to adapt to a physiological stressor. Robust differences in the transcriptome of wild type versus Akt1 null junctional zones were identified. Among the differentially expressed junctional zone transcripts was forkhead box O4 (Foxo4), which encodes a transcription factor and known AKT substrate. FOXO4 expression was prominent in the junctional zone and invasive trophoblast cells of the rat placentation site and enhanced following rat TS cell differentiation. Foxo4 gene disruption using genome-editing resulted in placentomegaly, including an enlarged junctional zone. AKT1 and FOXO4 regulate the expression of many of the same transcripts expressed by trophoblast cells; however, in opposite directions. In summary, we have identified AKT1 and FOXO4 as part of a regulatory network controlling hemochorial placenta development. Overall design: 1) Rat placentation site compartments were dissected on gd18.5. Dissected junctional zone tissues from wild type, Akt1 null, and Foxo4Xm- fetuses were snap-frozen in liquid nitrogen and stored at -80°C until processing for RNA isolation. 2) Foxo4 was knocked down in rat trophoblast stem cells using lentiviral delivery of shRNA. Following selection by puromycin, cells were differentiated for fifteen days. On day fifteen of differentiation, cells treated with either control shRNA or Foxo4-specific shRNA were collected and RNA was isolated.