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 RARα1−/−γ mice to excess retinol in vitro.
RARα1−/−γ+/− 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 μM) 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.
Whereas RARα1−/−γ+/+ and RARα1−/−γ+/− limbs exhibited deleterious effects on limb outgrowth and chondrogenesis in the presence of exogenous retinol, this outcome was significantly attenuated in RARα1−/−γ−/− limbs. The expressions of sox9 and col2a1 were significantly decreased in retinol-exposed RARα1−/−γ+/+ limbs. In contrast, expression was not altered in limbs from their RARα1−/−γ+/− or RARα1−/−γ−/− littermates. Retinol exposure upregulated the expression of meis1 and meis2 in RARα1−/−γ+/+ limbs; however, in RARα1−/−γ−/− limbs the expression of both genes was unresponsive to retinol. Pbx1a remained unresponsive to retinol treatment in all genotypes.
In the absence of RARα1, RARγ is a functionally important mediator of retinoid-induced limb dysmorphogenesis. Birth Defects Research (Part A), 2005. © 2005 Wiley-Liss, Inc.