Role of retinoic acid receptors alpha1 and gamma in the response of murine limbs to retinol in vitro.
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.
Galdones
Eugene
E
Department of Pharmacology and Therapeutics, McGill University, Montréal, Canada.
Lohnes
David
D
Hales
Barbara F
BF
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
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