Ransgenic GFP reporter /GFP/Rs1 mice vs. Col1/GFP mice) were

Ransgenic GFP reporter /GFP/Rs1 mice vs. Col1/GFP mice) were FACS sorted for GFP expression to separate two cell populations in each genotype. GFP-negative cells, representing various non-OB cell lineages mixed with osteoprogenitor and immature OBs, were clearly separated from the Exp Cell Res. Author manuscript; available in PMC 2016 May 01. Wattanachanya et al. Page 8 GFP-positive cell populations. Approximately 10% of the isolated cells are typically GFP positive. Sorted populations were 9799% pure by FACS reanalysis. Identifying GPCRs expressed on OBs and genes differentially expressed in Rs1 mice We compared the gene expression profiles in mature OBs from transgenic mice and controls from three biological replicates of each genotype. The identifiable cell populations were processed through RNA extraction, in vitro transcription, cDNA labeling and array hybridization. The Affymetrix Mouse Gene 1.0 ST Arrays were used to determine differences in gene expression. Each array contains a total of 26,166 coding transcripts. Significantly regulated genes were clustered using the GO Tree Machine, and functional analysis was done with GO-Elite software. We identified 358 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850363 differentially expressed transcripts if p 0.01 was set. Small magnitudes of differential expression were detected, ranging from 0.26-fold to 4.63-fold changes in expression. Applying a threshold of a greater than 1.5-fold change in expression level revealed a filtered list of 133 genes, 114 of which were associated with functional annotations in the GO-Elite software. The largest group of functionally related, differentially expressed genes was those involved in cell cycle and transcriptional regulation. Others included genes associated with apoptosis, enzyme activity, transporter and membrane protein. We observe no changes in genes associated with skeletal signaling pathways. Because our transgenic mouse model supported the concept of noncell-autonomous effects of OB Gs signaling, we sought to identify candidate paracrine mediators by determining the effect of Rs1 on the expression level of genes encoding secreted proteins. We identified 13 regulated genes, from those in Author Manuscript Author Manuscript Author Manuscript Author Manuscript Discussion We previously reported that strong basal Gs signaling by the Rs1 RASSL expressed in OBs driven by the 2.3-kb Col1 promoter fragment resulted in a dramatic age-dependent increase in endochondral bone formation. In this study, we investigated in detail the effects of increased Gs signaling in mature OBs on intramembranous bone formation in neonatal and growing Rs1 transgenic mice. The phenotype of Rs1 Scutellarein calvariae resembled that seen in Rs1 femoral bones, indicating that Gs signaling in mature OBs is MedChemExpress Sutezolid sufficient to initiate dramatic and similar skeletal responses in both endochrondral and intramembranous bones. The histological analyses demonstrated that Rs1 expression produces an expansion of osteoprogenitor cells that do not express Rs1, supporting the notion that the effect of Rs1 signaling in mature OBs alters bone formation by regulating the expression of factors that influence the early commitment, proliferation, and/or differentiation of osteoprogenitors. To determine the cellular response in vivo by which mature OBs regulate bone formation in response to Gs signaling, we utilized a microarray approach to examine Rs1-induced alterations in the OB transcriptome. The approach was used to identify an approximate snapshot of the.Ransgenic GFP reporter /GFP/Rs1 mice vs. Col1/GFP mice) were FACS sorted for GFP expression to separate two cell populations in each genotype. GFP-negative cells, representing various non-OB cell lineages mixed with osteoprogenitor and immature OBs, were clearly separated from the Exp Cell Res. Author manuscript; available in PMC 2016 May 01. Wattanachanya et al. Page 8 GFP-positive cell populations. Approximately 10% of the isolated cells are typically GFP positive. Sorted populations were 9799% pure by FACS reanalysis. Identifying GPCRs expressed on OBs and genes differentially expressed in Rs1 mice We compared the gene expression profiles in mature OBs from transgenic mice and controls from three biological replicates of each genotype. The identifiable cell populations were processed through RNA extraction, in vitro transcription, cDNA labeling and array hybridization. The Affymetrix Mouse Gene 1.0 ST Arrays were used to determine differences in gene expression. Each array contains a total of 26,166 coding transcripts. Significantly regulated genes were clustered using the GO Tree Machine, and functional analysis was done with GO-Elite software. We identified 358 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850363 differentially expressed transcripts if p 0.01 was set. Small magnitudes of differential expression were detected, ranging from 0.26-fold to 4.63-fold changes in expression. Applying a threshold of a greater than 1.5-fold change in expression level revealed a filtered list of 133 genes, 114 of which were associated with functional annotations in the GO-Elite software. The largest group of functionally related, differentially expressed genes was those involved in cell cycle and transcriptional regulation. Others included genes associated with apoptosis, enzyme activity, transporter and membrane protein. We observe no changes in genes associated with skeletal signaling pathways. Because our transgenic mouse model supported the concept of noncell-autonomous effects of OB Gs signaling, we sought to identify candidate paracrine mediators by determining the effect of Rs1 on the expression level of genes encoding secreted proteins. We identified 13 regulated genes, from those in Author Manuscript Author Manuscript Author Manuscript Author Manuscript Discussion We previously reported that strong basal Gs signaling by the Rs1 RASSL expressed in OBs driven by the 2.3-kb Col1 promoter fragment resulted in a dramatic age-dependent increase in endochondral bone formation. In this study, we investigated in detail the effects of increased Gs signaling in mature OBs on intramembranous bone formation in neonatal and growing Rs1 transgenic mice. The phenotype of Rs1 calvariae resembled that seen in Rs1 femoral bones, indicating that Gs signaling in mature OBs is sufficient to initiate dramatic and similar skeletal responses in both endochrondral and intramembranous bones. The histological analyses demonstrated that Rs1 expression produces an expansion of osteoprogenitor cells that do not express Rs1, supporting the notion that the effect of Rs1 signaling in mature OBs alters bone formation by regulating the expression of factors that influence the early commitment, proliferation, and/or differentiation of osteoprogenitors. To determine the cellular response in vivo by which mature OBs regulate bone formation in response to Gs signaling, we utilized a microarray approach to examine Rs1-induced alterations in the OB transcriptome. The approach was used to identify an approximate snapshot of the.