Perturbation of retinoic acid (RA)-mediated limb development suggests a role for diminished RA signaling in the teratogenesis of ethanol.
BACKGROUND
A proposed mechanism for ethanol teratogenicity entails ethanol-mediated reductions in retinoic acid (RA). This premise was investigated utilizing a mouse model, with limb reduction defects as the teratogenic end point.


METHODS
Ethanol, Disulfiram, or BMS-189453 was administered to C57BL/6J mice on the 9(th) day of pregnancy. Forelimb morphology was assessed on gestation day 18 using Alcian blue and Alizarin red staining. Nile blue sulfate or LysoTracker Red (LTR) vital staining identified cell death in the limb bud. The ability of RA to prevent ethanol-induced cell death was assessed by coadministration followed by laser scanning confocal microscopic examination of LTR-staining. In situ hybridization and qPCR were used to examine gene expression in treated limb buds.


RESULTS
Ethanol, Disulfiram, and BMS-189453 resulted in postaxial ectrodactyly, intermediate ectrodactyly, and other digital defects. Excessive Nile blue sulfate staining was evident in the presumptive AER following each of the three exposures. Ethanol-induced LTR staining was prevented by RA supplementation. Both in situ hybridization and qPCR illustrated decreases in Shh and Tbx5 in ethanol-exposed embryos as compared to control.


CONCLUSIONS
Contrary to studies of prolonged RA deficiency, acute exposure to functional antagonists of RA results in limb defects that are morphologically similar to those caused by ethanol. The rescue of ethanol-induced cell death by RA and similar changes in Shh transcription further suggest that RA contributes to ethanol-induced limb dysmorphology. Moreover, the repression of key mediators of limb development soon after ethanol exposure adds to the existing knowledge of the pathogenic effects of ethanol.
Johnson
Corey S
CS
The Department of Cell and Developmental Biology, and Bowles Center for Alcohol Studies, The University of North Carolina, Chapel Hill, North Carolina, USA.
Zucker
Robert M
RM
Hunter
Edward Sidney
ES
Sulik
Kathleen K
KK
D000014:Abnormalities, Drug-Induced / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D000431:Ethanol / Q000633:toxicity*; D005260:Female; D053823:Hedgehog Proteins / Q000235:genetics; D017403:In Situ Hybridization; D017880:Limb Deformities, Congenital / Q000139:chemically induced* / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020825:T-Box Domain Proteins / Q000235:genetics; D013723:Teratogens / Q000633:toxicity; D014212:Tretinoin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D053823:Hedgehog Proteins; D012333:RNA, Messenger; C465570:Shh protein, mouse; D020825:T-Box Domain Proteins; C103172:T-box transcription factor 5; D013723:Teratogens; D000431:Ethanol; D014212:Tretinoin