Gestational ethanol exposure disrupts the expression of FGF8 and sonic hedgehog during limb patterning

Ethanol is known to induce a wide variety of gestational anomalies, including skeletal malformations. Gestational ethanol exposure in mice has been shown to induce postaxial digit loss (ectrodactyly). How ethanol induces limb malformations is not understood. To better understand how ethanol effects limb development, we have utilized a transgenic line of mice that expresses beta-galactosidase in the apical ectodermal ridge (AER) of the limbs throughout gestation. METHODS: Pregnant female mice were injected with 2.9, 3.4, or 3.9 gm/kg ethanol at E9.3 and E9.5; embryos were isolated at E11.25, stained for beta-galactosidase activity, and evaluated for AER defects. Based upon the pattern of defects seen, expression of FGF8 in the AER and Sonic hedgehog in the postaxial mesoderm was evaluated by in situ hybridization. RESULTS: Two distinct phenotypes were seen in response to ethanol that were dose dependent. At 2.9 gm/kg ethanol, the most prevalent phenotype was a mislocalization of the AER to regions both dorsal and ventral to the midline. A higher dosage of 3.4 gm/kg ethanol did not increase the mislocalization phenotype, but resulted in a higher frequency of postaxial loss of the AER and associated mesenchymal tissue. The highest dosage utilized (3.9 gm/kg) resulted in a high frequency of both preaxial and postaxial loss of the AER. Through in situ hybridization, we found that ethanol exposure resulted in a concomitant reduction in FGF8 expression in the AER and Sonic hedgehog expression from the zone of polarizing activity (ZPA). CONCLUSIONS: We propose a model where ethanol disrupts the AER/ZPA positive feedback loop to induce postaxial malformations. Preaxial malformations seen at higher ethanol dosage suggest FGF8 as a critical target of ethanol in producing limb defects. (C) 2004 Wiley-Liss, Inc.