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.

Conclusion: In the absence of RARalpha1, RARgamma is a functionally important mediator of retinoid-induced limb dysmorphogenesis.