Thesis (M.Sc.) Uppsala : Sveriges lantbruksuniv.

Retinol (vitamin A) and its active metabolite, all-trans retinoic acid, signal through nuclear retinoic acid and retinoid X receptor (RAR/RXR) heterodimers. These complexes regulate the expression of genes involved in developmental processes such as limb development. In excess, retinoids are potent teratogens and cause marked reductive effects on the developing limb. The goal of this thesis was to elucidate the molecular mechanisms underlying retinoid-induced limb dysmorphogenesis. Specifically, using an in vitro limb culture system, I examined the involvement of one RAR isoform, RARgamma, in mediating retinoid insult. / My first objective was to examine how limbs deficient in RARgamma responded to exogenous retinoid exposure. I showed that RARgamma-null limbs (on an RARalpha1-null background) exhibited less severe limb defects following retinoid insult when compared to their wild-type counterparts. Additionally, the absence of RARgamma abolished the retinoid-induced misregulation of genes important for chondrogenesis (Sox9 and Col2a1 ) and limb outgrowth (Meis-1 and -2). / The next objective set out to determine how pharmacological activation of RARgamma affected limb development. The RARgamma-selective agonist (BMS-189961) caused limb dysmorphology (namely, effects on cartilage) that was comparable to pan-RAR activation with all-trans retinoic acid. A chondrogenesis-focused gene array analysis identified Mgp and Gdf10 as two RARgamma-responsive genes that may mediate retinoid-induced limb insult. / Subsequently, I assessed the functional involvement of Mgp in mediating retinoid teratogenicity. Limbs were treated with all- trans retinoic acid and warfarin (an inhibitor of MGP); warfarin co-treatment rescued limbs from retinoid-induced insult. / My final objective was to determine the importance of Gdf10 in mediating limb development. Recombinant human Gdf10-soaked beads were implanted into distal limb structures; ectopic overexpression of Gdf10 in the web (but not the digital ray) resulted in marked proximal limb malformations. / Collectively, these studies have illustrated the importance of RARgamma in retinoid teratology and have identified several potential mechanisms by which retinoids cause limb defects.

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