On was quantified as described in qPCR section. The results of the 
experiments were confirmed by repeating the experiment three times. Mineralization BMSCs cultures were performed in the absence or presence of murine Fgf9. BMSCs were isolated from WT mice, collected in PCM and plated onto 10cm cell culture dishes at a density of 7106 cells/dish. After incubating BMSCs in PCM for five days, the medium was removed along with all non-adherent cells and SCM was replaced and changed every two or three days. 5 ng/ml murine Fgf9 was added to the culture system from day 0 to day 20. To assess mineralization, two TPGS percent silver 
nitrate solution was added to cell culture dishes on day 20 for Von Kossa staining Exp Cell Res. Author manuscript; available in PMC 2016 May 01. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Wattanachanya et al. Page 7 and UV-crosslinked for 10 minutes. Stained cultures were scanned and quantified using Improvision Openlab software version 5.0.2. The results of the experiment were confirmed by repeating the experiment three times. Statistical analysis All qPCR data were evaluated using a two-tailed Student’s t tests assuming equal variance. Data were presented as the mean SD. Statistical significance was taken as p<0.05. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Results Bone histomorphometry Mice expressing Rs1 displayed notable asymmetric thickening of the calvariae starting at one week of age, and the observed phenotypes became more severe with age. By histological assessment, Rs1 calvariae had a dramatic increase in trabecular bone volume with a distorted cortical structure. High-magnification images of 9-week-old Rs1 calvariae revealed a large number of cells with stromal-type morphology between trabeculae with many appearing stacked on and near the trabecular surfaces. Bone marrow elements appeared to be scattered in small areas between the trabecular bones. Bone formation rates and patterns in 9-week-old samples were examined by dynamic histomorphometry. However, we could not quantify bone formation rate in the mutant mice due to the disorganized nature of bone formation, which displayed a diffuse labeling pattern consistent with the rapid bone formation. Bone immunohistochemistry Osterix expression was readily detected by immunohistochemistry in cells throughout the intertrabecular space in Rs1 calvariae, but not in controls. OBs that attached to cortical bone surfaces in control calvariae might be fully mature and express only low levels of Osterix. Osteocalcin was expressed predominantly in cells along bone surfaces in mutant and control mice. Immunohistochemistry with an antibody against the FLAG tag on Rs1 identified a significant number of cells within the Rs1 calvariae located along bone surfaces at the same location as Osteocalcin-expressing cells. They represented maturing OBs, based on the known expression patterns of the 2.3-kb Col I promoter fragment and on the abundant Osteocalcin in the bone lesions. Endothelial cells, identified by CD31 expression, were broadly distributed within the expanded calvarial tissue of Col1/Rs1 mice. The immunohistochemistry results indicated that many of the uniform cells in the mutant bone lesions are in the OB lineage with an increase in normal-looking blood vessel structures. Isolation of GFP-expressing cell population Cell populations obtained by enzymatic digestion of calvariae from 1-week-old 118414-82-7 22102576?report=abstract” title=View Abstract(s)”>PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850718,22102576 double and triple t.On was quantified as described in qPCR section. The results of the experiments were confirmed by repeating the experiment three times. Mineralization BMSCs cultures were performed in the absence or presence of murine Fgf9. BMSCs were isolated from WT mice, collected in PCM and plated onto 10cm cell culture dishes at a density of 7106 cells/dish. After incubating BMSCs in PCM for five days, the medium was removed along with all non-adherent cells and SCM was replaced and changed every two or three days. 5 ng/ml murine Fgf9 was added to the culture system from day 0 to day 20. To assess mineralization, two percent silver nitrate solution was added to cell culture dishes on day 20 for Von Kossa staining Exp Cell Res. Author manuscript; available in PMC 2016 May 01. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Wattanachanya et al. Page 7 and UV-crosslinked for 10 minutes. Stained cultures were scanned and quantified using Improvision Openlab software version 5.0.2. The results of the experiment were confirmed by repeating the experiment three times. Statistical analysis All qPCR data were evaluated using a two-tailed Student’s t tests assuming equal variance. Data were presented as the mean SD. Statistical significance was taken as p<0.05. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Results Bone histomorphometry Mice expressing Rs1 displayed notable asymmetric thickening of the calvariae starting at one week of age, and the observed phenotypes became more severe with age. By histological assessment, Rs1 calvariae had a dramatic increase in trabecular bone volume with a distorted cortical structure. High-magnification images of 9-week-old Rs1 calvariae revealed a large number of cells with stromal-type morphology between trabeculae with many appearing stacked on and near the trabecular surfaces. Bone marrow elements appeared to be scattered in small areas between the trabecular bones. Bone formation rates and patterns in 9-week-old samples were examined by dynamic histomorphometry. However, we could not quantify bone formation rate in the mutant mice due to the disorganized nature of bone formation, which displayed a diffuse labeling pattern consistent with the rapid bone formation. Bone immunohistochemistry Osterix expression was readily detected by immunohistochemistry in cells throughout the intertrabecular space in Rs1 calvariae, but not in controls. OBs that attached to cortical bone surfaces in control calvariae might be fully mature and express only low levels of Osterix. Osteocalcin was expressed predominantly in cells along bone surfaces in mutant and control mice. Immunohistochemistry with an antibody against the FLAG tag on Rs1 identified a significant number of cells within the Rs1 calvariae located along bone surfaces at the same location as Osteocalcin-expressing cells. They represented maturing OBs, based on the known expression patterns of the 2.3-kb Col I promoter fragment and on the abundant Osteocalcin in the bone lesions. Endothelial cells, identified by CD31 expression, were broadly distributed within the expanded calvarial tissue of Col1/Rs1 mice. The immunohistochemistry results indicated that many of the uniform cells in the mutant bone lesions are in the OB lineage with an increase in normal-looking blood vessel structures. Isolation of GFP-expressing cell population Cell populations obtained by enzymatic digestion of calvariae from 1-week-old PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850718,22102576 double and triple t.
