Retinoic acid receptor gamma-induced misregulation of chondrogenesis in the murine limb bud in vitro.
Vitamin A derivatives modulate gene expression through retinoic acid and rexinoid receptor (RAR/RXR) heterodimers and are indispensable for limb development. Of particular interest, RARgamma is highly expressed in cartilage, a target affected following retinoid-induced limb insult. The goal of this study was to examine how selective activation of RARgamma affects limb development. Forelimbs from E12.5 CD-1 mice were cultured for 6 days in the presence of all-trans RA (pan-RAR agonist; 0.1 or 1.0 microM) or BMS-189961 (BMS961, RARgamma-selective agonist; 0.01 or 0.1 microM) and limb morphology assessed. Untreated limbs developed normal cartilage elements whereas pan-RAR or RARgamma agonist-treated limbs exhibited reductive effects on chondrogenesis. Retinoid activity was assessed using RAREbeta2 (retinoic acid response element beta2)-lacZ reporter limbs; after 3 h of treatment, both drugs increased retinoid activity proximally. To elucidate the expression profiles of a subset of genes important for development, limbs were cultured for 3 h and cRNA hybridized to osteogenesis-focused microarrays. Two genes, matrix GLA protein (Mgp; chondrogenesis inhibitor) and growth differentiation factor-10 (Gdf10/Bmp3b) were induced by RA and BMS-189961. Real-time PCR was done to validate our results and whole mount in situ hybridizations against Mgp and Gdf10 localized their upregulation to areas of cartilage and programmed cell death, respectively. Thus, our results illustrate the importance of RARgamma in mediating the retinoid-induced upregulation of Mgp and Gdf10; determining their roles in chondrogenesis and cell death will help further unravel mechanisms underlying retinoid teratogenicity.
Galdones
Eugene
E
Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada.
Hales
Barbara F
BF
D000818:Animals; D020219:Chondrogenesis / Q000187:drug effects* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005552:Forelimb / Q000187:drug effects* / Q000196:embryology / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D017930:Genes, Reporter; D055413:Growth Differentiation Factor 10 / Q000378:metabolism; D018878:Limb Buds / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000819:agonists* / Q000378:metabolism; D015203:Reproducibility of Results; D020218:Response Elements / Q000187:drug effects; D012176:Retinoids / Q000633:toxicity*; D013723:Teratogens / Q000633:toxicity*; D046509:Tissue Culture Techniques; D014212:Tretinoin / Q000633:toxicity*; D000097768:Retinoic Acid Receptor gamma
D055413:Growth Differentiation Factor 10; D011506:Proteins; D018168:Receptors, Retinoic Acid; D012176:Retinoids; D013723:Teratogens; D014212:Tretinoin; D000097768:Retinoic Acid Receptor gamma; C514178:BMS 961; C525784:Gdf10 protein, mouse; C039391:granuloma protein, mouse