Gap Junctions Are Required for Trophoblast Proliferation in Early Human Placental Development * T. Nishimura a,b,1 , C. Dunk a,b,1 , Y. Lu a , X. Feng c , A. Gellhaus d , E. Winterhager d , J. Rossant a,b and S. J. Lye a,b,e, * a Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada; b Department of Physiology, University of Toronto, Toronto, Canada; c University Health Network, Toronto General Hospital Research Institute, Toronto, Canada; d Institute of Anatomy, University of Essen, Essen, Germany; e Department of Obstetrics and Gynecology, University of Toronto, Canada Paper accepted 7 January 2004 Little is known about the role of gap junctional intercellular communication (GJIC) in human trophoblast differentiation, particularly during the formation of extravillous trophoblast (EVT) cell columns and their subsequent differentiation into invasive cells. We have identified transcripts for five connexin gap junction proteins in the early human placenta (Cx32, Cx37, Cx40, Cx43 and Cx45). Of these, Cx40 and Cx45 proteins immunolocalize to EVT in anchoring cell columns. Cx40 expression is prominent in the anchoring column throughout the first trimester of pregnancy (6–14 weeks gestation). We used first trimester placental villous explant cultures to determine the functional significance of the inhibition of GJIC in EVT cell proliferation and differentiation using two known GJIC inhibitors, carbenoxolone (CBX) and heptanol. The morphology of EVT outgrowths changed dramatically upon GJIC-blockade, from compact and organized outgrowths into a scattered group of rounded individual trophoblast cells, reminiscent of an early invasive phenotype. Furthermore, the inhibition of GJIC in placental explants by CBX or heptanol induced a switch away from the proliferative and towards an invasive EVT phenotype, as evident from (a) the loss of the proliferation marker Ki67 and (b) an increase in the invasive marker a1 integrin. We also utilized antisense oligonucleotides to inhibit Cx40 protein expression in placental explants. Cx40 antisense treatment also resulted in the abolishment of outgrowth EVT cell proliferation (as determined by Ki67 immunostaining). Together, these results suggest that gap junctions composed particularly of Cx40 channels are required for the proliferation of EVT cells in anchoring cell columns, and that a loss of GJIC contributes to differentiation to the invasive EVT phenotype. Placenta (2004), 25, 595–607 Ó 2004 Elsevier Ltd. All rights reserved INTRODUCTION In the human placenta, specialized structures known as anchor- ing villi facilitate the attachment of the placenta to the uterine wall and provide the source of invasive cells that penetrate the maternal decidua and colonize maternal vessels. At the tips of the anchoring villi, the extravillous trophoblast (EVT) cells proliferate to form EVT cell columns. At the distal edge of the column, the EVT differentiate to become one of two popula- tions of invasive EVT: either interstitial trophoblast or endo- vascular trophoblast. The interstitial trophoblast invades through the decidual stroma as far as the superficial layer of the myometrium and appears to preferentially home towards the uterine spiral arteries and encircle them [1]. In contrast, the endovascular trophoblast arises as groups of trophoblast cells which detach from the trophoblastic shell and invade the lumen of the decidual uterine spiral arteries. The EVT mediate sub- stantial vascular remodeling, which ultimately results in vessel dilatation and an increase in blood flow to the intervillous space and placental transport interface (reviewed in Ref. [2]). Although EVT cells have been compared in their behaviour to malignant tumour cells [3,4], in contrast to tumourigenesis, the programmes of EVT proliferation and invasion are tem- porally separated resulting in two distinct proliferative and invasive EVT phenotypes [5]. Aberrant EVT proliferation and invasion can lead to pathological diseases, such as choriocar- cinoma (resulting from EVT hyperproliferation and over- invasion) or preeclampsia (resulting from inadequate EVT invasion) [2]. Investigations into the mechanisms that underlie cell column formation, migration, EVT differentiation, and invasion are extensive. Numerous proteins including basic * This work was supported by a grant from the NIH (HD42558-01) and by a fellowship from the Canadian Institutes of Health Research (to C.D.). 1 T. Nishimura and C. Dunk contributed equally to this manuscript. * Corresponding author. Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, 600 University Avenue, Toronto, ON, Canada, M5G 1X5. Tel.: +1-416-586-8258; fax: +1-416-586-8745. E-mail address: lye@mshri.on.ca (S.J. Lye). 0143–4004/$–see front matter Ó 2004 Elsevier Ltd. All rights reserved Placenta (2004), 25, 595–607 doi:10.1016/j.placenta.2004.01.002