Molecular mechanisms of Vitamin A-induced limb defects in the mid-organogenesis-stage mouse limb bud

The goal of this thesis was to move toward defining the molecular mechanism(s) of Vitamin A-induced limb malformations. Vitamin A and its biologically active metabolites, the retinoids, are potent limb teratogens, inducing reductive defects, preceded by decreased chondrogenesis and upregulated apoptosis. To target the origins of these effects, we focused initially on characterizing the pathway of Vitamin A-induced apoptosis. Our first objective was to identify mediators of the degradation and commitment phases of this process. We show dose-dependent activation of the effector caspase, caspase-3, and increased mitochondrial cytochrome-c release after treatment. The next objective was to determine whether these changes were receptor-mediated. Both pan-RAR (BMS 189453) and, surprisingly, pan-RXR (HX603) antagonists were able to ameliorate Vitamin A-induced limb malformations and apoptosis, indicating both receptor subtypes are important mediators of these processes. Interestingly, exposure to either antagonist alone also upregulated apoptosis. Each compound induced a specific, reproducible pattern of limb bud apoptosis, reiterating the fact that proper regulation of this process is crucial for normal limb morphology. Our final objective was to identify genes that may be responsible for the apoptosis and other pathologies observed after retinoid exposure. Vitamin A treatment significantly upregulated 81 genes, including key limb development signaling molecules, oncogenes, extracellular matrix/cell adhesion molecules, and transcriptional regulators such as Id3, Snai1, Hes1 and Eya2. To link these expression changes to teratogenic outcome, the response of these 4 genes was assessed after Vitamin A exposure, and after limb bud rescue with BMS 189453 and HX603; expression levels were correlated with limb malformations. Pathway analysis of the significantly upregulated genes reveals that many that are associated with cell-cycle, apoptosis and chondrogenesis are functionally linked. Moreover, members of these cascades cross-talk with one another, indicating that retinoids affect multiple cellular processes in a coordinated fashion to induce their teratogenic effects. Collectively, these studies have advanced our understanding of Vitamin A-mediated apoptotic pathways. In addition, we have proposed several mechanisms for retinoid-induced limb malformations, identified novel retinoid targets, and revealed multiple avenues for future research