The function and interrelationship between GDF5 and ERG-010 during chondrogenesis in vitro Mark Howard 1,2,3 & Rocky S. Tuan 2 & Gillian A. Wallis 1 Received: 26 June 2015 /Accepted: 15 September 2015 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2015 Abstract Joint formation begins with the establishment of an interzone within the cartilaginous anlagen of the future skel- eton. Both GDF5 and ERG are proposed as regulators of chondrocyte differentiation during and post interzone forma- tion. The aim of this study was to examine the relationship between Gdf5 and Erg expression and downstream effects on chondrocyte gene expression. Erg expression was identified in mouse knee joints at E13.5. Expression analyses were per- formed using micromass cultures of murine C3H10T1/2 mes- enchymal cells undergoing induced chondrogenesis in the presence and absence of GDF5 and ERG. At E13.5, Erg ex- pression was found to surround epiphyseal chondrocytes and span the interzone up to the intermediate zone. Erg splice forms were expressed in micromass cultures, and their expres- sion profile was altered by the addition of recombinant GDF5 depending on the stage of differentiation. Overexpression of Erg-010 resulted in a downregulation of Col2a1 and Col10a1. Microarray analysis following Erg-010 overexpression iden- tified two potential downstream targets, Ube2b and Osr2, which were also differentially regulated by GDF5. Erg regu- lation by GDF5 in induced mesenchymal cells in vitro is de- pendent on the stage of chondrogenesis, and its expression in vivo demarcates chondrocytes that are not destined to be consumed by endochondral ossification. Functionally, Erg ex- pression causes downregulation of Col2a1 and Col10a1 ex- pression and this effect is potentially mediated by Osr2 and/or Ube2b. Combined, these data suggest a possible pathway linking GDF5, ERG and downstream factors in the processes of chondrocyte differentiation during articular joint formation. Keywords Chondrogenesis . Articularcartilage . Erg . Gdf5 . Endochondral ossification Introduction Synovial joints are composed of specialised tissues encased in a joint capsule that enables articulation be- tween two opposing skeletal elements. These tissues in- clude articular cartilage, bone, ligament, synovium and a fibrous capsule (Archer et al. 2003). During embryogen- esis, a continuous cartilage anlagen is laid down in the developing limb at the site of the future skeletal ele- ments; within this, joints develop at specified positions. In the mouse, at E12.5, there is no morphological sign of the future knee joint location until, at E13.5, chondrocytes of the anlagen flatten, elongate to form the joint interzone, switch off collagen type II expression and commence collagen type I expression. Thereafter, the interzone is the major signalling centre of joint formation Electronic supplementary material The online version of this article (doi:10.1007/s11626-015-9960-5) contains supplementary material, which is available to authorized users. * Mark Howard mark.howard@sbcna.com * Rocky S. Tuan rst13@pitt.edu * Gillian A. Wallis g.wallis@manchester.ac.uk 1 Wellcome Trust Centre for Cell Matrix Research, University of Manchester, Manchester M13 9PL, UK 2 Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, School of Medicine, University of Pittsburgh, 450 Technology Drive, Room 221, Pittsburgh, PA 15219, USA 3 Centre for Endocrinology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-015-9960-5