Homeobox gene Distal-less 3 (DLX3) is a regulator of villous cytotrophoblast differentiation A. Chui a, b , D.A. Evseenko c , S.P. Brennecke a, b , J.A. Keelan d , B. Kalionis a, b , P. Murthi a, b, * a Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Australia b Department of Obstetrics and Gynaecology, University of Melbourne, Parkville, Victoria 3052, Australia c Department of Pathology and Laboratory Medicine, University of California Los Angeles (UCLA), Los Angeles, CA, USA d School of Women’s and Infants’ Health University of Western Australia, King Edward Memorial Hospital, Subiaco, Western Australia 6008, Australia article info Article history: Accepted 7 July 2011 Keywords: Placenta DLX3 Homeobox Function abstract Dlx3, a member of the large homeobox gene family of transcription factors, is important for murine placental development. Targeted deletion of Dlx3 in the mouse results in embryonic death due to placental failure. This study investigated the role of human DLX3 in villous cytotrophoblast (VCT) differentiation in the placenta. Primary VCT from human term placentae, which spontaneously differ- entiate when maintained in culture over 72 h, showed a significant increase in mRNA and protein expression of DLX3 and 3bHSD. The functional role of DLX3 was determined using trophoblast derived- cell line, BeWo. Forskolin treated BeWo cells showed significantly increased DLX3 mRNA and protein expression. Forskolin stimulation also showed a significant increase in syncytin and 3bHSD mRNA expression, and increased release of bhCG into the cell culture supernatant. To determine whether DLX3 had a direct or indirect effect on VCT differentiation, mRNA and protein expression of DLX3 was increased using a plasmid DLX3 over-expression construct. Over-expression of DLX3 resulted in increased mRNA expression of 3bHSD and syncytin, as well as increased secretion of b-hCG protein in the cell culture medium. In conclusion, we provide evidence that DLX3 acts upstream of syncytin, 3bHSD and bhCG and that DLX3 has a regulatory role in VCT differentiation. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction The placenta is a structural and physiological barrier between the fetal and maternal circulations, providing an exchange interface for nutrients, gases and wastes [1]. During early human pregnancy, mononuclear villous cytotrophoblast cells fuse to form a multinu- cleated syncytium. For continual exchange, the syncytiotrophoblast layer must grow and be refreshed by differentiation and fusion of underlying, proliferating villous cytotrophoblast cells. It is impor- tant that villous cytotrophoblast differentiation is tightly controlled because inadequate differentiation would compromise the production and function of the syncytiotrophoblast and reduce the efficiency of the maternal-fetal exchange system. Villous cytotrophoblast differentiation is accompanied by increased expression of critical hormones and factors such as the human chorionic gonadotropin b-subunit (b-hCG), a marker of in vitro differentiation [2], 3b-hydroxysteroid dehydrogenase (3bHSD), an enzyme essential for the maintenance of pregnancy [3], and syncytin-1, a retroviral envelope protein that appears to be primarily expressed in the syncytiotrophoblast [4]. Villous cytotrophoblast cell functions are regulated by a variety of growth factors, cytokines and transcription factors [5e10]. This study focuses on a member of the large family of transcription factors, called the homeobox genes. Homeobox genes play impor- tant roles in mammalian embryonic development [11] and are characterised by a conserved 60 amino acid homeodomain which is necessary for DNA binding [12,13]. Mouse knockout studies of homeobox genes provide evidence for critical roles of these regu- latory genes in placental development [14e16]. Our specific interest lies in a sub-family of homeobox genes called Distal-less. Members of the Distal-less gene (Dlx) organised in pairs as 1e6 on chromosome 17q21, play important roles in cellular differenti- ation [17e20]. Targeted deletion of mouse Dlx3 results in embry- onic death by day 10 due to placental vascularisation defects that alter the development of the labyrinth, which is thought to be equivalent in functions to the syncytiotrophoblast in humans [21,22]. Furthermore, Dlx3 is involved in 3bHSD placental hormone * Corresponding author. University of Melbourne, Department of Obstetrics and Gynaecology, Royal Women’s Hospital, Parkville, Victoria 3052, Australia. Tel.: þ613 8345 3747; fax: þ613 8345 3746. E-mail address: padma@unimelb.edu.au (P. Murthi). Contents lists available at ScienceDirect Placenta journal homepage: www.elsevier.com/locate/placenta 0143-4004/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2011.07.007 Placenta 32 (2011) 745e751