Microarray on osteoblasts made from bone marrow stromal cells from Bmp2 flox/flox and Bmp2; Prx1-Cre mice

Enhanced BMP or canonical Wnt (cWnt) signaling are therapeutic strategies employed to enhance bone formation and fracture repair, but the mechanisms each pathway utilizes to specify cell fate of bone-forming osteoblasts remain poorly understood. Among all BMPs expressed in bone, we find that singular deficiency of Bmp2 blocks the ability of cWnt signaling to specify osteoblasts from limb bud or bone marrow progenitors. When exposed to cWnts, Bmp2-deficient cells fail to progress through the Runx2/Osx1 checkpoint and thus do not upregulate multiple genes controlling mineral metabolism in osteoblasts. Cells lacking Bmp2 after induction of Osx1 differentiate normally in response to cWnts, supporting pre-Osx1+ osteoprogenitors as a critical source and target of BMP2. Our analysis furthermore reveals Grainyhead-like 3 (Grhl3) is to date an unidentified transcription factor in the osteoblast gene regulatory network that is induced during bone development and bone repair, and acts upstream of Osx in a BMP2-dependent manner. The Runx2/Osx1 transition therefore receives critical regulatory inputs from BMP2 that are not compensated for by cWnt signaling, and this is mediated at least in part by induction and activation of Grhl3. Gene level differential expression analysis was performed with Affymetrix Mouse ST 2.1 microarrays on bone marrow stromal cells from Bmp2F/F or Bmp2Prx1Δ/Δ mice that were differentiated for 7 days in osteogenic medium alone, plus BMP2 (100 ng/ml), plus Wnt3a (40 ng/ml), or BMP2 plus Wnt3a. Cultures were performed in biological triplicates using pooled cells from n≥3 mice/genotype. Traditional cDNA, generated from 1 ug total RNA using EcoDry Premix (Clonetech), was used for pre-microarray and post-microarray validation by QPCR. Eight cDNA libraries were made from 100 ng total RNA using WT Expression Kit (Ambion): 1 pooled sample from 3 independent cultures of Bmp2F/F cells grown in osteogenic medium (33.33 ng each for a total of 100 ng); 1 pooled sample from 3 independent cultures of Bmp2Prx1Δ/Δ cells + BMP2 + Wnt3a (33.33 ng each for a total of 100 ng); 3 samples from 3 independent cultures of Bmp2F/F cells + Wnt3a (100 ng each); and 3 samples from 3 independent cultures of Bmp2Prx1Δ/Δ cells + Wnt3a (100 ng each). Data was analyzed with Expression Console and Transcriptome Analysis Console v3.0 (free from Affymetrix). Gene expression was reported as linear fold change relative to Bmp2Prx1Δ/Δ cells + Wnt3a, and p-value was calculated by one-way between-subject ANOVA for unpaired samples.