TOXICOLOGICAL SCIENCES 90 1 208 220 2006 doi 10 1093 toxsci kfj060 Advance Access publication December 1 2005 Retinoid Receptor Antagonists Alter the Pattern of Apoptosis in Organogenesis Stage Mouse Limbs Sarah E Ali Khan and Barbara F Hales1 Department of Pharmacology and Therapeutics McGill University Montre al Que bec Canada H3G 1Y6 Received September 2 2005 accepted November 21 2005 Exposure of murine limbs in vitro to vitamin A retinol induces limb reduction defects and apoptosis To assess the relative roles of the retinoic acid receptors RARs and the retinoid X receptors RXRs embryonic day 12 murine limbs were cultured with selective RAR or RXR antagonists in the presence or absence of teratogenic concentrations of retinol Both antagonists alone impaired limb development in the presence of teratogenic concentrations of retinol both attenuated limb malformations Abnormal limb morphology whether caused by excessive or attenuated retinoid signaling by retinol or either antagonist respectively was correlated with increased apoptosis after 24 h of drug exposure We conclude that in the developing limb antagonists selective for either member of the RAR RXR heterodimer attenuate retinoid signaling and block the teratogenic signaling of excess retinol Improvements in limb morphology in the presence of either the RAR or the RXR antagonist coincided with restoration of the extent and localization of limb bud apoptosis to control patterns Key Words retinoic acid limb development limb malformations teratogen Vitamin A or retinol and its derivatives the retinoids are essential for proper embryonic development Active retinoids are important signaling molecules in the regulation of cell differentiation proliferation and morphogenesis the developing limb is sensitive to both excess and paucity of these compounds Genetic and biochemical manipulations indicate that retinoids are crucial for vertebrate limb development at two stages in early organogenesis for forelimb bud initiation and later in mid organogenesis for proper limb outgrowth Mic et al 2004 Niederreither et al 2002 In the mouse and other model species administration of excess bioactive retinoids during mid organogenesis leads to reductive limb defects resembling those seen in retinoid deficiency truncations and deletions of 1 To whom correspondence should be addressed at Department of Pharmacology and Therapeutics McGill University 3655 Promenade Sir WilliamOsler Montre al Que bec Canada H3G 1Y6 Fax 514 398 7120 E mail barbara hales mcgill ca the long bones ulna radius and humerus in the forelimb and digital deletions and fusions Kochhar 1973 1985 During this susceptible period key morphogenetic processes occurring in the developing limb bud include limb outgrowth differentiation of the limb skeleton cartilaginous anlagen and the selective deletion of specific cells by programmed cell death or apoptosis 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 of apoptosis in shaping limbs during development we have examined the effects of retinoids on this event in the organogenesis stage murine limb Limb bud apoptosis in the mouse begins about embryonic day ED 10 5 peaking at ED14 5 with the majority occurring in the interdigital INZ anterior and posterior marginal zones AMZ and PMZ and the apical ectodermal ridge AER Zakeri and Ahuja 1997 Precise spatiotemporal control of both the extent and the localization of apoptosis during development is essential for normal morphogenesis exposure of the embryo to excessive retinol during mid organogenesis coincident with the induction of limb malformations also causes aberrant apoptosis Retinoids bind to two subclasses of nuclear receptors the retinoic acid receptors RARs and the retinoid X receptors RXRs each consisting of three members a b and c Leid et al 1992 Several isoforms of each exist with distinct spatial and temporal expression patterns The diverse effects of retinoids during normal development are mediated by the RAR RXR heterodimer made up of various combinations of these isoforms Chambon 1994 Kastner et al 1997 Alltrans and 9 cis retinoic acid bind and activate transcription from the RARs while just 9 cis retinoic acid binds RXRs with high affinity On ligand binding the heterodimers transactivate through DNA response element recognition In vivo studies following administration of selective retinoid receptor agonists show that RARa selective agonists are the most potent limb teratogens followed by b and c specific agonists Arafa et al 2000 Conversely RXR selective agonists are not fetotoxic or teratogenic when administered in vivo Kochhar et al 1996 but administration of these compounds can potentiate the teratogenic effects of an The Author 2005 Published by Oxford University Press on behalf of the Society of Toxicology All rights reserved For Permissions please email journals permissions oxfordjournals org RETINOID RECEPTOR ANTAGONISTS AND APOPTOSIS RAR selective agonist Elmazar et al 2001 In sum the presence of both the RAR and the RXR subtypes is important in the limb and their relationships with one another in the heterodimer are complex Yet little is known about how these receptors influence apoptosis in normal development or during the evolution of abnormal morphology We had two goals in the present work 1 to investigate the relationship between retinoid induced limb malformations and apoptosis and 2 to assess the importance of the RARs and the RXRs in this process To do this we targeted retinoid signaling during mid organogenesis with retinoid receptor antagonists first in the absence of exogenous ligand endogenous situation and second in the presence of teratogenic concentrations of retinol Enzymes metabolizing retinol to both all trans and 9 cis retinoic acid are present in the limb Horton and Maden 1995 thus generating ligands for both the RAR and RXR receptor subfamilies ED12 limb buds were treated in an in vitro culture system using selective pan RAR BMS 453 and panRXR HX603 antagonists on their own as well as in combination with exogenous all trans retinol acetate Vitamin A MATERIALS AND METHODS Limb bud cultures and drug treatments Limb buds were cultured as previously described Ali Khan and Hales 2003 Huang and Hales 2002 Briefly ED12 embryos were dissected from timed pregnant CD1 mice Charles River Canada Inc St Constant QC At this stage embryos were 5 6 mm in