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, RAR gamma 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 RAR gamma 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 mu M) or BMS-189961 (BMS961, RAR gamma-selective agonist; 0.01 or 0.1 mu M) and limb morphology assessed. Untreated limbs developed normal cartilage elements whereas pan-RAR or RAR gamma agonist-treated limbs exhibited reductive effects on chondrogenesis. Retinoid activity was assessed using RARE beta 2 (retinoic acid response element beta 2)-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 RAR gamma 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.