Homeobox gene distal-less 3 is expressed in proliferating and differentiating cells of the human placenta A. Chui a, b , N.A. Pathirage a, b , B. Johnson f , M. Cocquebert c, d, e , T. Fournier c, d, e , D. Evain-Brion c, d, e , B. Roald g , U. Manuelpillai h , S.P. Brennecke a, b , B. Kalionis a, b , P. Murthi a, b, * a Department of Perinatal Medicine, Pregnancy Research Centre, The Royal Women’s Hospital, Parkville, Victoria 3052, Australia b Department of Obstetrics and Gynaecology, The University of Melbourn, Parkville, Victoria 3052, Australia c INSERM, U767, France d Université Paris Descartes, France e PremUP Foundation, Faculté des Sciences Pharmaceutiques et Biologiques, Paris F 75006, France f Division of Hematology, IMVS, Hanson Institute, Adelaide, SA 5000, Australia g Center for Clinical Research, University of Oslo, Oslo, Norway h Monash Institute of Medical Research, Department of Medicine, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia article info Article history: Accepted 11 May 2010 Keywords: Placenta Homeobox genes Gene expression Protein expression Distal-less 3 Trophoblast cells abstract 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, syn- cytiotrophoblast 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 tropho- blasts. 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. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction The development of a functional placenta is critical for a successful pregnancy. It is the site of feto-maternal transport of gases, nutrients and metabolites. Trophoblast stem cells give rise to two specific cytotrophoblast lineages, villous cytotrophoblasts and extravillous cytotrophoblasts. In the villous trophoblast, a fusion phenotype, the trophoblast differentiates from the fusion of mononuclear cytotrophoblastic cells into a syncytium, the syncy- tiotrophoblast. Bathing the maternal blood, the syncytiotropho- blast is involved in maternal-fetal exchanges and in placental endocrine functions. In the extravillous trophoblast, a proliferative and an invasive phenotype, the cytotrophoblastic cells proliferate and migrate as individual cells, and are involved in the interstitial invasion of the endometrial decidua and myometrium and remodeling of uterine spiral arteries [1]. This study focuses on homeobox transcription factors in human placental development. Homeobox genes play important roles in mammalian embryonic development [2,3] and are characterised by a conserved 60 amino acid homeodomain which is necessary for DNA binding [4,5]. Several mouse knockout studies of homeobox genes provide evidence for critical roles of these regulatory genes in placental development. For example, targeted deletion of Cdx4, Cdx2 and Esx1 cause severe and diverse morphological, vascular and angiogenesis defects of the placenta [6e8]. Furthermore, the downregulation of the homeobox gene HOXA11 may be essential for the fusion of cytotrophoblasts into syncytiotrophoblast [9]. Our specific interest is in a sub-family of homeobox genes called the Distal-less homeobox genes. The Distal-less homeobox gene * Corresponding author. Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women’s Hospital, Parkville, Victoria 3052, Australia. Tel.: þ61 3 8345 3747; fax: þ61 3 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 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.05.003 Placenta 31 (2010) 691e697