Full Length Article Loss of ephrinB1 in osteogenic progenitor cells impedes endochondral ossification and compromises bone strength integrity during skeletal development Thao M. Nguyen a,b , Agnieszka Arthur a,b,d,1 , Sharon Paton a,b,c , Sarah Hemming a,b , Romana Panagopoulos a , John Codrington e , Carl R. Walkley f , Andrew C.W. Zannettino b,c , Stan GronthosProfessor a,b, ⁎ ,1 a Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia b South Australian Health and Medical Research Institute, Adelaide, SA, Australia c Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia d Division of Haematology, SA Pathology, Adelaide, SA, Australia e School of Mechanical Engineering, University of Adelaide, Adelaide, Australia f St Vincent's Institute of Medical Research and Department of Medicine, St Vincent's Hospital, University of Melbourne, Fitzroy, Vic, Australia abstract article info Article history: Received 17 June 2016 Revised 16 August 2016 Accepted 9 September 2016 Available online 10 September 2016 The EphB receptor tyrosine kinase family and their ephrinB ligands have been implicated as mediators of skeletal development and bone homeostasis in humans, where mutations in ephrinB1 contribute to frontonasal dysplasia and coronal craniosynostosis. In mouse models, ephrinB1 has been shown to be a critical factor mediating oste- oblast function. The present study examined the functional importance of ephrinB1 during endochondral ossifi- cation using the Cre recombination system with targeted deletion of ephrinB1 (EfnB1 fl/fl ) in osteogenic progenitor cells, under the control of the osterix (Osx:Cre) promoter. The Osx:EfnB1 -/- mice displayed aberrant bone growth during embryonic and postnatal skeletal development up to 4 weeks of age, when compared to the Osx:Cre con- trols. Furthermore, compared to the Osx:Cre control mice, the Osx:EfnB1 -/- mice exhibited significantly weaker and less rigid bones, with a reduction in trabecular/ cortical bone formation, reduced trabecular architecture and a reduction in the size of the growth plates at the distal end of the femora from newborn through to 4 weeks of age. The aberrant bone formation correlated with increased numbers of tartrate resistant acid phosphatase pos- itive osteoclasts and decreased numbers of bone lining osteoblasts in 4 week old Osx:EfnB1 -/- mice, compared to Osx:Cre control mice. Taken together, these observations demonstrate the importance of ephrinB1 signalling be- tween cells of the skeleton required for endochondral ossification. © 2016 Elsevier Inc. All rights reserved. Keywords: Eph/ephrin Osterix Endochondral ossification Osteoprogenitors Osteoclasts Chondrocytes 1. Introduction The Eph receptor tyrosine kinase (RTK) family consists of Eph recep- tors and ephrin ligands which can be divided into two subclasses based structure and binding affinity. The ephrin (Efn) ligands are GPI-tethered (A-subclass) or transmembrane (B-subclass) molecules. Unlike most RTKs, both Eph and ephrin molecules can mediate cell signalling. For- ward signalling is achieved through receptor classes, while reverse sig- nalling is mediated through the ephrin ligand leading to different biological outcomes (reviewed by [1]). These contact-dependent, cell membrane-associated molecules can mediate inhibitory, repulsive and attractive cellular responses, and are involved in numerous develop- mental and post-natal biological processes and pathologies. The Eph/ ephrin molecules, predominately known for their role in establishing and maintaining cellular migration [2,3] and boundary formation [4,5] can also stimulate cellular differentiation [6,7]. Pivotal studies by Zhao et al. [7] demonstrated the importance of the EphB family in mouse bone homeostasis. Their studies showed that ac- tivation of EphB4 signalling in osteoblasts promoted mineral formation, while reverse signalling through the EphB4 cognate ligand, ephrinB2, inhibited osteoclast maturation and function [7]. EphB/ephrinB interac- tions via EphB4/ephrinB2 and EphB2/ephrinB have been found to medi- ate cell attachment, migration and osteo/chondrogenic differentiation of human bone marrow derived mesenchymal stem cell (MSC) [8,9]. More recently, we and others have postulated that ephrinB1 may be a key regulator of skeletal development and bone homeostasis [8–12].A global mouse knockout and numerous human mutations of ephrinB1 re- sult in cranial defects such as frontonasal dysplasia and coronal cranio- synostosis [13–15]. The global deletion of ephrin-B1 in mouse also Bone 93 (2016) 12–21 Abbreviations: Efn, ephrin; Osx, osterix; fl, floxed; MSC, mesenchymal stem cells; HSC, hematopoietic stem cells. ⁎ Corresponding author at: Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, 5005, South Australia, Australia. E-mail address: stan.gronthos@adelaide.edu.au (S. Gronthos). 1 Co-senior authors. http://dx.doi.org/10.1016/j.bone.2016.09.009 8756-3282/© 2016 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Bone journal homepage: www.elsevier.com/locate/bone