br Results br Discussion In

Results
Discussion In this work, we confirm that pnPRX1+ AMG-517 of the calvaria presented gene expression profiles of SSC\'s, are localized to the calvarial suture niche, and are required for calvarial bone regeneration. Global ablation strategies of SSCs do not allow to unequivocally conclude that individual locations (i.e., the sutures) represent their unique niches because absence of the cells from other locations may be responsible for the observed effects. We evaluated the contribution of pnPRX1+ cells of the sutures to calvarial bone regeneration in three different settings (global ablation, regional suturectomy, and orthotopic suture transplantation). This triple experimental strategy allowed us to confirm that sutures are the exclusive niches of the calvarial SSCs expressing PRX1. Our work demonstrates that expression of PRX1 identifies a subpopulation of the pnGLI1+ cells identified by Zhao et al. (2015). Interestingly, global ablation of pnPRX1+ cells does not result in a major developmental phenotype whereas global ablation of pnGLI1+ cells averts craniofacial growth and induces craniosynostosis. This is expected for two reasons: First, pnPRX1+ cells are discretely distributed across the suture space and their ablation should therefore generate a phenotype less significant than the one obtained by ablation of the widely distributed pnGLI1+ cells. Second, preosteoblasts and osteoblasts of the calvaria have life spans longer than 60 days (Park et al., 2012) and represent a reservoir of cells able to form bone even in absence of their progenitors (in our case pnPRX1+ cells). Thus, the effects of the ablation of SSCs should be seen in long-term studies and may influence craniofacial development only if ablation is performed early enough during development, when a significant pool of SSCs is required. Indeed, when global ablation of PRX1-expressing cells is performed during embryogenesis, we observe an incomplete calvarial bone formation. Maruyama et al. (2016) did not perform global ablation studies of pnAXIN2+ cells. However, the fact that postnatal OSTERIX-expressing cells co-express AXIN2 (Tan et al., 2014) and the fact that Osx is expressed in preosteoblasts (Nakashima et al., 2002; Strecker et al., 2013) suggests that ablation of pnAXIN2+ cells could generate a more significant development phenotype than the one observed in the pnPRX1+ cell ablation studies. Although gene knockout studies cannot be correlated to ablation studies of cells expressing the same genes, the fact that knockout of Axin2 generates premature suture closing and craniosynostosis (Yu et al., 2005) suggests that this may in fact be the case. Our studies indicate that pnPRX1+ cells express low levels of Axin2 and that these levels increase upon WNT agonist stimulation. This result may indicate that either pnPRX1+ cells are the same cells identified by Maruyama et al. (pnAXIN2+ cells) or that pnPRX1+ cells are a subpopulation of pnAXIN2+ cells. Since, at least in long bones, the majority of the postnatal bone cells expressing OSX co-express AXIN2 (Tan et al., 2014), pnPRX1+ cells differ from the OSX-expressing cells of the suture (Figure 1E), and ablation of pnPRX1+ does not interfere with postnatal calvarial development, we speculate that pnPRX1+ cells are a subpopulation of the pnAXIN2+ cells identified by Maruyama et al. Additional studies are required to test this postulation. In conclusion, our studies showed that pnPRX1+ cells are responsible for calvarial bone regeneration and represent an initial step toward future investigations aiming to identify novel, effective therapies for calvarial malformations and defects.
Experimental Procedures
Author Contributions
Acknowledgments We acknowledge Dr. Shunichi Murakami (Case Western Reserve University) for kindly providing the Prx1-creER-EGFP mice, Dr. David Scadden (Harvard Stem Cell Institute) for providing the Col1-EGFP mice, Dr. Dolrudee Jumlongras and Dr. Bjorn Olsen (Harvard School of Dental Medicine) for providing the Gli1-creER; tdTOMATO mice, and Dr. Jenna Galloway (Massachusetts General Hospital) for sharing the collagenase-based cell isolation protocol. We thank Matthew Warman, Steven Hann, and Mark Kahan (Warman Lab at Children\'s Hospital of Boston) for their assistance with the preliminary Prx1-driven DTA ablation studies. We also thank Ronald Mathieu, Mahnaz Paktinat, and Yanhui Deng at the Children\'s Hospital & Harvard Stem Cell Institute Flow Cytometry Research Facility (Karp Research Labs, 1 Blackfan Circle, Boston) for their kind cooperation. We thank John R. Martin at the HSDM Micro CT Core Facility (188 Longwood Avenue, REB 316, Boston) and Daniel Brooks and Michael Armanini at the Harvard/MGH Center for Skeletal Research, Imaging and Biomechanical Testing Core for their technical assistance. This project was supported by grant no. R00DE021069 (NIH/NIDCR) to G.I. and by the HSDM Dean’s Scholar Award to L.J.M. and S.-C.A.Y.