title: Role of retinoic acid receptors alpha1 and gamma in the response of murine limbs to retinol in vitro.
abstract: BACKGROUND
  Derivatives of retinol (vitamin A), commonly referred to as retinoids, signal through retinoic acid and retinoid X receptors (RARs/RXRs) and are essential for normal limb formation. Retinoid imbalances or perturbations in receptor function result in aberrant limb development. To examine the mechanisms underlying retinol-induced limb defects, we determined the responsiveness of limbs from RARalpha1-/-gamma mice to excess retinol in vitro.
  
  
  METHODS
  RARalpha1-/-gamma+/- mice were bred and their embryos were recovered at gestational day (GD) 12.5. The forelimbs were excised and cultured in vitro in the presence of all-trans retinol acetate (0, 1.25, 12.5, or 62.5 microM) for 6 days. The expression profiles of genes known to affect chondrogenesis (sox9 and col2a1) and limb outgrowth (meis1, meis2, and pbx1a) were examined by real-time qRT-PCR following retinol exposure for 3 hr.
  
  
  RESULTS
  Whereas RARalpha1-/-gamma+/+ and RARalpha1-/-gamma+/- limbs exhibited deleterious effects on limb outgrowth and chondrogenesis in the presence of exogenous retinol, this outcome was significantly attenuated in RARalpha1-/-gamma-/- limbs. The expressions of sox9 and col2a1 were significantly decreased in retinol-exposed RARalpha1-/-gamma+/+ limbs. In contrast, expression was not altered in limbs from their RARalpha1-/-gamma+/- or RARalpha1-/-gamma-/- littermates. Retinol exposure upregulated the expression of meis1 and meis2 in RARalpha1-/-gamma+/+ limbs; however, in RARalpha1-/-gamma-/- limbs the expression of both genes was unresponsive to retinol. Pbx1a remained unresponsive to retinol treatment in all genotypes.
  
  
  CONCLUSIONS
  In the absence of RARalpha1, RARgamma is a functionally important mediator of retinoid-induced limb dysmorphogenesis.
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montréal, Canada.
authors.forename: Eugene
authors.initials: E
authors.lastname: Galdones
authors.forename: David
authors.initials: D
authors.lastname: Lohnes
authors.forename: Barbara F
authors.initials: BF
authors.lastname: Hales
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D020219:Chondrogenesis / Q000187:drug effects; D024043:Collagen Type II / Q000378:metabolism; D005121:Extremities / Q000196:embryology; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D005865:Gestational Age; D006609:High Mobility Group Proteins / Q000378:metabolism; D017880:Limb Deformities, Congenital / Q000139:chemically induced*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009924:Organ Culture Techniques; D011247:Pregnancy; D018168:Receptors, Retinoic Acid / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D000072482:Retinoic Acid Receptor alpha; D055758:SOX9 Transcription Factor; D013723:Teratogens / Q000633:toxicity*; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D014801:Vitamin A / Q000633:toxicity*; D000097768:Retinoic Acid Receptor gamma
chemical_list: D024043:Collagen Type II; D006609:High Mobility Group Proteins; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D055758:SOX9 Transcription Factor; D013723:Teratogens; D014157:Transcription Factors; D014801:Vitamin A; D000097768:Retinoic Acid Receptor gamma; C447413:Col2a1 protein, mouse; C000607072:Rara protein, mouse; C527933:Sox9 protein, mouse