RESEARCH ARTICLE Precise spatial restriction of BMP signaling in developing joints is perturbed upon loss of embryo movement Pratik Narendra Pratap Singh 1, *, Claire A. Shea 2 , Shashank Kumar Sonker 1 , Rebecca A. Rolfe 2 , Ayan Ray 1, ‡ , Sandeep Kumar 1 , Pankaj Gupta 1 , Paula Murphy 2,§ and Amitabha Bandyopadhyay 1,§ ABSTRACT Dynamic mechanical loading of synovial joints is necessary for normal joint development, as evidenced in certain clinical conditions, congenital disorders and animal models where dynamic muscle contractions are reduced or absent. Although the importance of mechanical forces on joint development is unequivocal, little is known about the molecular mechanisms involved. Here, using chick and mouse embryos, we observed that molecular changes in expression of multiple genes analyzed in the absence of mechanical stimulation are consistent across species. Our results suggest that abnormal joint development in immobilized embryos involves inappropriate regulation of Wnt and BMP signaling during definition of the emerging joint territories, i.e. reduced β-catenin activation and concomitant upregulation of pSMAD1/5/8 signaling. Moreover, dynamic mechanical loading of the developing knee joint activates Smurf1 expression; our data suggest that Smurf1 insulates the joint region from pSMAD1/5/8 signaling and is essential for maintenance of joint progenitor cell fate. KEY WORDS: Wnt/BMP signaling, Articular cartilage, Immobilization, Joint development, Muscle contraction, Mechanoregulation, Mechanosensitivity INTRODUCTION We have previously proposed that joint development requires spatially appropriate specification and differentiation of bi-potential cartilage cells, which take on one of two distinct fates (Ray et al., 2015). The cells at and near the joint line are exposed to canonical Wnt signaling and assume permanent or articular cartilage fate, whereas the cells at a distance from the interzone are exposed to BMP signaling and differentiate as transient cartilage, eventually being replaced by bone cells. It is now well established that dynamic mechanical movement of limbs is not only important for maintenance of articular cartilage in adults but also for embryonic articular cartilage differentiation (Rolfe et al., 2013). Loss of movement during joint development in either embryonic chicken or mouse model systems results in ectopic development of transient cartilage across the presumptive joint region, characterized by Alcian Blue staining, downregulation of joint specific markers and ectopic expression of an early marker of transient cartilage, Col2a1, with continuous cartilage formation across the joint site in extreme cases (Kahn et al., 2009; Nowlan et al., 2010; Roddy et al., 2011). Although the early phase of joint specification, including formation of the interzone, is not affected, tissue patterning within the presumptive joint and maintenance of the articular territory is lost. Little is known about the molecular mechanisms that are perturbed upon immobilization and lead to loss of articular cartilage cells. Using both in vitro and in vivo model systems, many genes have been identified whose expression levels change upon alteration of the mechanical environment (Bougault et al., 2012; Dowthwaite et al., 1999; Kahn et al., 2009; Roddy et al., 2011; Rolfe et al., 2014a,b; Sironen et al., 2002a,b). The importance of the Wnt pathway in mechanoregulation of joint development was shown by altered expression of multiple Wnt pathway genes in muscle-less mouse embryos (Rolfe et al., 2014b) and reduced canonical pathway read- out in the developing joints of a reporter mouse line (Kahn et al., 2009). A series of prior studies demonstrated that canonical Wnt signaling is crucial for articular cartilage differentiation and maintenance (Guo et al., 2004; Hartmann and Tabin, 2001; Kahn et al., 2009; Ray et al., 2015; Später et al., 2006a,b). However, although knockout of Wnt ligands/β-catenin leads to misexpression of Col2a1 in the embryonic joint region, unlike the loss of mechanical stimulation, articular cartilage markers continue to be expressed (Später et al., 2006a), suggesting that decreased canonical Wnt signaling is not the only mechanistic explanation for failure to maintain joint progenitor cells in immobile embryos. In several studies, both in chick and mouse, ectopic activation of BMP signaling in the putative interzone/articular cartilage cells led to ectopic expression of Col2a1 and absence of articular joints (Ray et al., 2015; Zou et al., 1997) – a phenotype strikingly similar to immobilization. We therefore investigated the possibility of misregulation of BMP signaling in the absence of dynamic mechanical loading of joints and possible interplay between BMP and Wnt signaling in the mechanoresponsive definition and maintenance of the joint territory. This possibility was further supported by the identification of altered BMP pathway gene expression in muscle-less embryos (Rolfe et al., 2014b; R.A.R and P.M., unpublished). Here, by comparing gene expression patterns in decamethonium bromide (DMB)-induced immobilized chick embryos, as well as in muscle-less mouse embryos, we demonstrate that molecular changes in and around developing joints upon immobilization are highly conserved. Investigating the mechanistic basis, we show downregulation of canonical Wnt pathway activity with concomitant upregulation of Sfrp2, combined with upregulation of BMP signaling in both chick and mouse embryos. We further show that expression of Smurf1, an intracellular inhibitor of BMP signaling, is not maintained in the absence of dynamic mechanical Received 24 April 2017; Accepted 9 February 2018 1 Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Uttar Pradesh 208016, India. 2 Department of Zoology, School of Natural Sciences, Trinity College Dublin, Dublin 2, Ireland. *Present address: Department of Medical Oncology and Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA 02215, USA. ‡ Present address: Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. § Authors for correspondence (abandopa@iitk.ac.in; paula.murphy@tcd.ie) P.N.P.S., 0000-0001-6830-2177; C.A.S., 0000-0002-7549-2711; S.K.S., 0000- 0002-0874-1642; R.A.R., 0000-0003-2177-3136; A.R., 0000-0002-7171-1737; P.M., 0000-0002-8048-6850; A.B., 0000-0002-0429-438X 1 © 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev153460. doi:10.1242/dev.153460 DEVELOPMENT