Retinoid Receptor Antagonists Alter the Pattern of Apoptosisin Organogenesis Stage Mouse LimbsSarah E. Ali-Khan and Barbara F. Hales1Department of Pharmacology and Therapeutics, McGill University, Montre´al, Que´bec, Canada H3G 1Y6Received September 2, 2005; accepted November 21, 2005Exposure of murine limbs in vitro to vitamin A (retinol) induceslimb reduction defects and apoptosis. To assess the relative roles ofthe retinoic acid receptors (RARs) and the retinoid X receptors(RXRs), embryonic-day-12 murine limbs were cultured withselective RAR or RXR antagonists in the presence or absence ofteratogenic concentrations of retinol. Both antagonists aloneimpaired limb development; in the presence of teratogenicconcentrations of retinol, both attenuated limb malformations.Abnormal limb morphology, whether caused by excessive orattenuated retinoid signaling by retinol or either antagonist,respectively, was correlated with increased apoptosis after 24 hof drug exposure. We conclude that, in the developing limb,antagonists selective for either member of the RAR/RXR hetero-dimer attenuate retinoid signaling and block the teratogenicsignaling of excess retinol. Improvements in limb morphology inthe presence of either the RAR or the RXR antagonist coincidedwith restoration of the extent and localization of limb budapoptosis to control patterns.Key Words: retinoic acid; limb development; limb malforma-tions; teratogen.Vitamin A or retinol and its derivatives, the retinoids, areessential for proper embryonic development. Active retinoidsare im portant signaling molecules in the regulation of celldifferentiation, prol iferation, and morphogenesis; the develop-ing limb is sensitive to both excess and paucity of thesecompounds.Genetic and biochemical manipulations indicate that reti-noids are crucial for vertebrate limb development at two stages,in early organogenesis for forelimb bud initiation, and later inmid-organogenesis, for proper limb outgrowth (Mic et al.,2004; Niederreither et al., 2002) In the mouse and other modelspecies, administration of excess bioactive retinoids duringmid-organogenesis leads to reductive limb defects resemblingthose seen in retinoid deficiency: truncations and dele tions ofthe long bones (ulna, radius, and humerus in the forelimb) anddigital deletions and fusions (Kochhar, 1973, 1985). Duringthis susceptible period, key morphogenetic processes occurringin the developing limb bud include limb outgrowth, differen-tiation of the limb skeleton cartilaginous anlagen, and theselective deletion of specific cells by programmed cell death orapoptosis. Retinoids influence all three of these processes(Dupe et al., 1999; Francis-West and Tickle, 1996; Kochhar,1985; Zakeri and Ahuja, 1994), but due to the importance ofapoptosis in shaping limbs during development, we haveexamined the effects of retinoids on this event in theorganogenesis-stage murine limb.Limb bud apoptosis in the mouse begins about embryonicday (ED) 10.5, peaking at ED14.5, with the majority occurringin the interdigital (INZ), anterior and posterior marginal zones(AMZ and PMZ) and the apical ectodermal ridge (AER)(Zakeri and Ahuja, 1997). Precise spatiotemporal control ofboth the extent and the localiza tion of apoptosis duringdevelopment is essential for normal morphogenesis; exposureof the embryo to excessive retinol during mid-organogenesis,coincident wi th the induction of limb malfo rmations, alsocauses aberrant apoptosis.Retinoids bind to two subclasses of nuclear receptors, theretinoic acid receptors (RARs) and the retinoid X receptors(RXRs), each consisting of three members: a, b, and c (Leidet al., 1992). Several isoforms of each exist, with distinctspatial and temporal expression patterns. The diverse effects ofretinoids during normal development are mediated by theRAR/RXR heterodimer made up of various combinations ofthese isoforms (Chambon, 1994; Kastner et al., 1997). All-trans and 9-cis retinoic acid bind and activate transcriptionfrom the RARs, while just 9-cis retinoic acid binds RXRs withhigh affinity. On ligand binding, the heterodimers transactivatethrough DNA response element recognition.In vivo studies following administration of selective retinoidreceptor agonists show that RARa-selective agonists are themost potent limb teratogens, followed by b- and c-specificagonists (Arafa et al., 2000). Conversely, RXR-selectiveagonists are not fetotoxic or teratogenic when administeredin vivo (Kochhar et al., 1996), but administrat ion of thesecompounds can potentiate the teratogenic effects of an1To whom correspondence should be addressed at Department of Pharma-cology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montre´al, Que´bec, Canada H3G 1Y6. Fax: (514) 398–7120. E-mail:[email protected].Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For Permissions, please email: [email protected] SCIENCES 90(1), 208–220 (2006)doi:10.1093/toxsci/kfj060Advance Access publication December 1, 2005RAR-selective agonist (Elmazar et al., 2001). In sum, thepresence of both the RAR and the RXR subtypes is importantin the limb, and their relationships with one another in theheterodimer are complex. Yet little is known about how thesereceptors influence apoptosis in normal development or duringthe evolution of abnormal morp hology.We had two goals in the present work: (1) to investigate therelationship between retino id-induced limb malformations andapoptosis and (2) to assess the importance of the RARs and theRXRs in this process. To do this, we targeted retinoid signalingduring mid-organogenesis with retinoid receptor antagonists,first in the absence of exogenous ligand (endogenous situation),and second in the presence of teratogenic concentrations ofretinol. Enzymes metabolizing retinol to both all-trans and9-cis retinoic acid are present in the limb (Horton and Maden,1995), thus generating ligands for both the RAR and RXRreceptor subfamilies. ED12 limb buds were treated in an in vitroculture system using selective pan-RAR (BMS 453) and pan-RXR (HX603) antagonists on their own, as well as in com-bination with exogenous all-trans retinol acetate (Vitamin A).MATERIALS AND METHODSLimb bud cultures and drug treatments. Limb buds were cultured aspreviously described (Ali-Khan and Hales, 2003; Huang and Hales, 2002).Briefly, ED12 embryos were dissected from timed-pregnant