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Human idiopathic fetal growth restriction (FGR) is associated with placental insufficiency. Previously, we reported that the expression of homeobox gene Distal-less 3 (DLX3) is increased in idiopathic FGR placentae and is a regulator of villous trophoblast differentiation. Here, we identify the downstream targets of DLX3 in trophoblast-derived cell lines. We modelled the high levels of DLX3 in FGR using an over-expression plasmid construct and complemented this using short-interference RNA (siRNA) for inactivation in cultured cells. Using a real-time PCR-based gene profiling, candidate target genes of DLX3 over-expression and inactivation were identified as regulators of trophoblast differentiation; GATA2 and PPARγ. The expression of GATA2 and PPARγ were further assessed in placental tissues and showed increased mRNA and protein levels in FGR-affected tissues compared with gestation-matched controls. We conclude that DLX3 orchestrates the expression of multiple regulators of trophoblast differentiation and that expression of these regulatory genes is abnormal in FGR.

Abnormal trophoblast function is associated with human fetal growth restriction (FGR). Targeted disruption of homeobox gene transforming growth β-induced factor (TGIF-1) results in placental dysfunction in the mouse. The role of human TGIF-1 in placental cell function is unknown. The aims of this study were to determine the expression of TGIF-1 in human idiopathic FGR-affected placentae compared with gestation-matched controls (GMC), to elucidate the functional role of TGIF-1 in trophoblasts and to identify its downstream targets. Real-time PCR and immunoblotting revealed that TGIF-1 mRNA and protein expression was significantly increased in FGR-affected placentae compared with GMC (n = 25 in each group P < 0.05). Immunoreactive TGIF-1 was localized to the villous cytotrophoblasts, syncytiotrophoblast, microvascular endothelial cells and in scattered stromal cells in both FGR and GMC. TGIF-1 inactivation in BeWo cells using two independent siRNA resulted in significantly decreased mRNA and protein of trophoblast differentiation markers, human chorionic gonadotrophin (CGB/hCG), syncytin and 3β-hydroxysteroid dehydrogenase/3β-honest significant difference expression. Our data demonstrate that homeobox gene TGIF-1 is a potential up-stream regulator of trophoblast differentiation and the altered TGIF-1 expression may contribute to aberrant villous trophoblast differentiation in FGR.

The placenta provides critical transport functions between the maternal and fetal circulations during intrauterine development. Formation of this interface is controlled by nuclear transcription factors including homeobox genes. Here we summarize current knowledge regarding the expression and function of homeobox genes in the placenta. We also describe the identification of target transcription factors including PPARγ, biological pathways regulated by homeobox genes and their role in placental development. The role of the nuclear receptor PPARγ, ligands and target genes in human placental development is also discussed. A better understanding of these pathways will improve our knowledge of placental cell biology and has the potential to reveal new molecular targets for the early detection and diagnosis of pregnancy complications including human fetal growth restriction.

Human idiopathic foetal growth restriction (FGR) is frequently associated with placental insufficiency. In our previous studies, we have reported the isolation and characterisation of the homeobox gene Distal-less 3 (DLX3) in the human placenta. In this study, we have investigated the level of DLX3 expression in idiopathic FGR-affected placentae and determined its functional role in villous trophoblast differentiation. FGR-affected placentae (n = 25) were collected based on well-defined clinical criteria and matched for gestation with control uncomplicated pregnancies (n = 25). Real-time polymerase chain reaction and immunoblotting showed increased DLX3 mRNA and protein expression in FGR-affected placentae compared with gestation-matched controls. Qualitative immunohistochemistry revealed DLX3 localisation in the syncytiotrophoblast, cytotrophoblasts and endothelial cells surrounding the foetal capillaries in both FGR-affected and control placentae. Down-regulation of DLX3 in primary villous trophoblast cells and a trophoblast-derived cell line showed decreased expression of differentiation markers, 3βHSD, βhCG and syncytin. Therefore, we conclude that increased DLX3 expression in FGR may contribute to trophoblast dysfunction observed in FGR.

DLX3, a member of the large homeobox gene family of transcription factors, is necessary for normal placentation. Targeted deletion of dlx3 in mouse resulted in embryonic death due to placental failure. This study demonstrates the presence of DLX3 mRNA expression in human first trimester and term placental tissue, cultured trophoblast-like cell lines and in isolated primary villous and extravillous trophoblast cells. Using an ovine polyclonal antibody, the spatial distribution was identified for DLX3 in human placental tissues, trophoblast cell lines and in freshly isolated primary trophoblast cells. A 50 kDa immunoreactive DLX3 protein was detected in the human placenta, in trophoblast cell lines and in primary trophoblast cells. Nuclear expression for DLX3 was observed in villous cytotrophoblasts, syncytiotrophoblast and extravillous cytotrophoblast in the proximal regions of the cytotrophoblast cell columns in first trimester placental tissues. Immunoreactivity was also detected in few stromal cells and microvascular endothelial cells surrounding the fetal capillaries. In the first trimester placental bed, DLX3 expression was predominantly observed in the cytoplasm of the endovascular and interstitial trophoblasts. We conclude that the cellular expression of DLX3 was extensive in the human placenta and propose that DLX3 may play an important role in normal placental development.