Calvarial bone phenotype at 1 and 9 weeks of age and investigated the

Calvarial bone phenotype at 1 and 9 weeks of age and investigated the effect of enhanced OB Gs signaling on the OB transcriptome by examining alterations in gene expression in vivo in OBs from calvariae of 1-week-old Rs1 mice, compared to controls. The functionally related, differentially expressed genes, the Gs- and Gi-GPCRs, and candidate paracrine mediators that may contribute to the observed skeletal phenotype of the effect of Gs signaling in OBs were determined. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Methods Transgenic mice To examine the influence of Gs signaling in OBs, we used a mutated version of the Gscoupled 5HT-4 serotonin receptor that has constitutive Gs signaling activity, together with the tetracycline-regulated system to regulate the expression of Rs1 in vivo. The Col1-tTA/TetO-Rs1 double transgenic mice /Rs1) were generated by crossing mice carrying the heterozygous TetO-Rs1 transgene with mice carrying the homozygous Col1-tTA transgene. Endochondral bones of these mutant mice were well characterized as described. In this study, we utilized green fluorescent protein -based reporters to identify Rs1 expressing cells PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850363 for investigating OB transcriptome analysis. We co-expressed a histoneGFP marker in OBs in vivo alone /GFP) or with Rs1 in triple transgenic mice /GFP/Rs1). This was accomplished by generating mice harboring a TetO-histone-GFP gene 47Efu/J, Jackson Dehydroxymethylepoxyquinomicin site Laboratory) with a TetO-Rs1 gene. Double transgenic mice were then crossed with mice homozygous for the 2.3 Col1-tTA genes to examine the skeletal effects of Gs signaling in mature OBs. Both transgenes driven by TetO were expressed in OBs only when the 2.3 Col1-tTA transgene was present. In this approach, 25% of the progeny were controls Exp Cell Res. Author manuscript; available in PMC 2016 May 01. Wattanachanya et al. Page 4 and 25% were experimental. All animals were maintained in the FVB/N background. Mice were on regular chow without doxycycline administration to allow transgene expression in experimental mice since conception. All protocols were approved by the Institutional Animal Care and Use Committee of the San Francisco Veterans Affairs Medical Center. Calvarial bone histology Calvariae collected for hematoxylin/eosin staining and immunohistochemistry were fixed in 10% neutral buffered formalin for 24 hours and stored at 4C in 70% ethanol. Bones were decalcified in 10% EDTA at 4C for 23 days BAY-41-2272 custom synthesis before paraffin embedding, sectioning, and H&E staining. Assessment of calvarial bone was performed on 10-m-thick sections at the midpoint of the parietal part. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Bone immunohistochemistry Osteocalcin and Osterix were detected on formalin-fixed, paraffin-embedded sections with primary antibodies from Takara Bio and AbCam as described. Since Rs1 bears an N-terminal FLAG tag, the detection of Rs1 expressing OBs was done on alcohol-fixed, paraffin-embedded bone sections with a M1 FLAG primary antibody in M.O.M. diluents. Detection of CD31, the endothelial cell marker, was performed using an Anti-Ig HRP detection kit following the manufacturer’s protocol. Static and dynamic histomorphometry Calvariae from 1- and 9-week-old mice were isolated at the time of euthanasia and fixed in 10% NBF for 12 days before embedding in methyl methacrylate and sectioning with Jung 2065 and 2165 microtomes. Sectioned bones were processed for Von Kossa staining as described. For dynamic.Calvarial bone phenotype at 1 and 9 weeks of age and investigated the effect of enhanced OB Gs signaling on the OB transcriptome by examining alterations in gene expression in vivo in OBs from calvariae of 1-week-old Rs1 mice, compared to controls. The functionally related, differentially expressed genes, the Gs- and Gi-GPCRs, and candidate paracrine mediators that may contribute to the observed skeletal phenotype of the effect of Gs signaling in OBs were determined. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Methods Transgenic mice To examine the influence of Gs signaling in OBs, we used a mutated version of the Gscoupled 5HT-4 serotonin receptor that has constitutive Gs signaling activity, together with the tetracycline-regulated system to regulate the expression of Rs1 in vivo. The Col1-tTA/TetO-Rs1 double transgenic mice /Rs1) were generated by crossing mice carrying the heterozygous TetO-Rs1 transgene with mice carrying the homozygous Col1-tTA transgene. Endochondral bones of these mutant mice were well characterized as described. In this study, we utilized green fluorescent protein -based reporters to identify Rs1 expressing cells PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850363 for investigating OB transcriptome analysis. We co-expressed a histoneGFP marker in OBs in vivo alone /GFP) or with Rs1 in triple transgenic mice /GFP/Rs1). This was accomplished by generating mice harboring a TetO-histone-GFP gene 47Efu/J, Jackson Laboratory) with a TetO-Rs1 gene. Double transgenic mice were then crossed with mice homozygous for the 2.3 Col1-tTA genes to examine the skeletal effects of Gs signaling in mature OBs. Both transgenes driven by TetO were expressed in OBs only when the 2.3 Col1-tTA transgene was present. In this approach, 25% of the progeny were controls Exp Cell Res. Author manuscript; available in PMC 2016 May 01. Wattanachanya et al. Page 4 and 25% were experimental. All animals were maintained in the FVB/N background. Mice were on regular chow without doxycycline administration to allow transgene expression in experimental mice since conception. All protocols were approved by the Institutional Animal Care and Use Committee of the San Francisco Veterans Affairs Medical Center. Calvarial bone histology Calvariae collected for hematoxylin/eosin staining and immunohistochemistry were fixed in 10% neutral buffered formalin for 24 hours and stored at 4C in 70% ethanol. Bones were decalcified in 10% EDTA at 4C for 23 days before paraffin embedding, sectioning, and H&E staining. Assessment of calvarial bone was performed on 10-m-thick sections at the midpoint of the parietal part. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Bone immunohistochemistry Osteocalcin and Osterix were detected on formalin-fixed, paraffin-embedded sections with primary antibodies from Takara Bio and AbCam as described. Since Rs1 bears an N-terminal FLAG tag, the detection of Rs1 expressing OBs was done on alcohol-fixed, paraffin-embedded bone sections with a M1 FLAG primary antibody in M.O.M. diluents. Detection of CD31, the endothelial cell marker, was performed using an Anti-Ig HRP detection kit following the manufacturer’s protocol. Static and dynamic histomorphometry Calvariae from 1- and 9-week-old mice were isolated at the time of euthanasia and fixed in 10% NBF for 12 days before embedding in methyl methacrylate and sectioning with Jung 2065 and 2165 microtomes. Sectioned bones were processed for Von Kossa staining as described. For dynamic.