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Administration of CdSO(4) to C57BL/6 mice at day 9.5 of gestation induces a high incidence of postaxial forelimb ectrodactyly in the offspring. We propose that Cd(2+) exposure impairs the process of anterior/posterior formation in the limb bud, a process that is directed by Sonic hedgehog (Shh) signaling. We show that exposure of the mouse embryo to Cd(2+) disrupts Shh signaling as measured by polarizing activity of mouse limb bud ZPA grafted to a host chick wing, and activity of a Gli:luciferase reporter exposed to limb bud lysates. Yet the expression of Shh and its translation are not affected by Cd(2+) exposure. We propose that teratogen exposure affects the processing of Shh in the cells in which it is made.

A unique limb phenotype is described in a radiation-induced mutant mouse resulting from an inversion of a proximal segment of chromosome 5. The limb phenotype in the homozygous mutant presents with two anterior skeletal elements in the zeugopod but no posterior bone, hence the name replicated anterior zeugopod, raz. The zeugopod phenotype is accompanied by symmetrical central polydactyly of hand and foot. The chromosomal inversion includes the Shh gene and the regulatory locus, located approximately 1 Mb away, within the Lmbr1 gene. In homozygous mutants, the expression of Shh mRNA and Shh protein is severely downregulated to about 20% of wild-type limb buds, but Shh expression appears normal throughout the remainder of the embryo. Correspondingly, Gli3 expression is upregulated and posteriorly expanded in the raz/raz limb bud. We propose that the double anterior zeugopod and symmetrical central polydactyly are due to an increased and uniform concentration of the Gli3 repressor form because of lowered Shh signaling.

We studied the relationship between the sidedness of visceral organs and the expression of limb abnormalities in the legless mutant. The control of asymmetry in visceral development appears to be random in the legless mutant; that is, 50% develop normally (situs solitus) and 50% develop with inverted viscera (situs inversus). We find that the sidedness of forelimb abnormalities expressed in the mutants is highly correlated with visceral sidedness. Abnormalities are more severe in the right forelimbs in situs solitus mutants, while the left forelimb is more severely affected in situs inversus mutants.

Exposure of C57BL/6J mice to three anticonvulsant derivatives, namely, dimethadione, sodium valproate, and sodium diphenylhydantoin, each induced postaxial forelimb ectrodactyly. The agents were administered at gestational days 9, 9 1/3, 9 2/3, and 10. It was determined that administration at day 9 2/3 induced the highest percentage of forelimb ectrodactyly for each of the three agents. The forelimb ectrodactyly response in the C57BL/6J strain was compared with the A/J strain (Collins et al., Teratology, 41:61-70, 1990); it was found that the C57BL/6J strain was more sensitive to dimethadione and the A/J strain was more sensitive to diphenylhydantoin and sodium valproate. The position of vertebral defects induced by sodium valproate correlated with the time of drug administration. The overall syndrome of malformations induced by the three anticonvulsant agents was relatively similar in the two mouse strains and differed between each of the anticonvulsant agents.

The effects of acetazolamide on the developing rodent limb bud were postulated to result from a reduction of intracellular pH (pHi). Embryonic intracellular pH was calculated from transplacental distribution of the weak acid, 5,5'-dimethyloxazolidine-2,4-dione, in teratogenically sensitive (C57BL/6) and resistant (SWV) inbred mice. pHi was reduced by acetazolamide treatment in C57 embryos and limb buds but not in SWV samples. Acetazolamide teratogenesis can be exacerbated by coadministration of amiloride, presumably through inhibition of Na+/H+ exchange attributable to the latter agent. pHi reduction after such treatment was more profound than after acetazolamide alone, providing further support for the central hypothesis. pH was also reduced in other embryonic (embryo plasma) and extraembryonic compartments (exocoelomic fluid, amniotic fluid). pH changes in these compartments could also lead or contribute to abnormal development.

It is now thought that ethanol exerts many of its behavioral effects in the CNS by interaction with the gamma-aminobutyric acid (GABA) receptor, and it has been shown that the benzodiazepine reverse agonist Ro15-4513 reverses some of the CNS effects produced by ethanol. The hypothesis was tested that ethanol exerts its teratogenic effects through interaction with a putative embryonic GABA receptor by determining whether Ro15-4513 reverses ethanol-induced forelimb ectrodactyly in C57BL/6 mice. First, pregnant C57BL/6 dams were injected twice i.p. with ethanol (2.9 g/kg body weight, 4 hr apart) on day 10 of gestation: 49% of the fetuses were resorbed or dead and 46% of the survivors showed forelimb ectrodactyly. In contrast, when SWV mice were treated with ethanol, embryolethality was only 11.9% and no forelimb ectrodactyly was observed. In a second experiment, when ethanol (2.6 g/kg x 2) was administered to C57BL/6 mice, 34% resorptions and 31% forelimb ectrodactyly were observed. Ectrodactyly induced by ethanol was primarily of the forelimb and exclusively postaxial. Ethanol produced an unusual forelimb defect in a small number of instances where there was a postaxial autopod reduction defect coupled with a preaxial zeugopod reduction defect. Ro15-4513 administered alone (50 mg/kg x 2) was neither embryolethal nor teratogenic in C57BL/6 mice. To attempt to reverse the teratogenic effect of ethanol, dams that were injected 5 min before each ethanol administration with Ro15-4513 (0.5, 1, 2.5, 5, 10 mg/kg twice) showed no significant change in frequency of forelimb ectrodactyly compared to embryos treated with ethanol alone. However, resorptions increased significantly to 77% and 62% with the 5 and 10 mg/kg doses of Ro15-4513. Thus there appears to be an embryolethal interaction of Ro15-4513 with ethanol. Nevertheless, since Ro15-4513 did not reverse the teratogenic effect induced by ethanol, these results do not support the hypothesis that the teratogenic mechanism of ethanol is mediated through a putative embryonic GABA receptor.

Exposure of A/J mice on day 9.5 of gestation to the derivatives of three acidic anticonvulsant agents, namely dimethadione, sodium valproate, and sodium diphenylhydantoin, each induced postaxial forelimb ectrodactyly predominantly of the right side. This specific malformation has previously been associated with the administration of acetazolamide to rodents; however, several agents can induce this same defect including other carbonic anhydrase inhibitors, carbon dioxide, cadmium, ethanol, ammonium chloride, and 13-cis retinoic acid. The relative potency of the three agents indicates no direct relationship to the pKa of the acid. Other than ectrodactyly, each of the anticonvulsant agents induced a compound-specific spectrum of malformations despite the uniform administration time. This finding suggests that these agents are capable of acting via different mechanisms or by the differential spatial and temporal dynamics of a common mechanism.

Exposure of C57BL/6J mice to 20% CO2 for 8 hours on day 10 of gestation has been shown to produce right-sided postaxial forelimb ectrodactyly in 23% of the offspring. Carbon dioxide exposure produces a dramatic increase in maternal plasma CO2 accompanied by an inevitable decrease in plasma pH, both of which appear to be involved in the induction of ectrodactyly. However, the low incidence of ectrodactyly associated with NH4Cl-induced metabolic acidosis suggests that the primary teratogenic factor in respiratory acidosis is elevated CO2 tension. This conclusion is supported by the observation that moderation of maternal plasma pH in the face of sustained elevated PCO2 fails to reduce the incidence of ectrodactyly; moreover, there is a strong correlation between maternal serum CO2 content and the incidence of ectrodactyly.

Exposure of C57BL/6J mice to CO2 during a critical period of gestation results in predominantly right-sided, postaxial, forelimb ectrodactyly in the offspring. The incidence and severity of CO2-induced limb malformations has been shown to be dependent on the concentration of inspired CO2, the developmental age of the embryo at exposure and the duration of CO2 exposure. Offspring of acetazolamide-treated C57BL/6J mice also display this highly specific form of ectrodactyly (Green et al., '73). Since the drug has been shown to elevate tissue CO2 tension (Mithoefer and Davis, '58), the teratogenic effect of acetazolamide may be related to induction of a hypercapnic embryonic environment.

We have attempted to elucidate the mechanism of cadmium teratogenesis utilizing inbred mouse strains sensitive (C57BL/6J) or resistant (SWV) to the embryotoxic effect of this common heavy metal contaminant. Carbonic anhydrase activity of whole-embryo homogenates was moderately depressed in C57BL/6J mice compared to a slight and transient decrease in the resistant SWV mice. Embryonic erythrocytes were similarly examined, and the cadmium did not have any effect on carbonic anhydrase activity in either strain. Likewise, histochemical examination of carbonic anhydrase activity did not reveal any effect of cadmium in the embryos of their strain. Generally, the zinc concentration of embryos was not affected by cadmium administration. However, increased levels of zinc were observed in cadmium-exposed yolk sacs of both strains suggesting that cadmium produces an adverse effect on yolk sac function. Untreated C57BL/6J units (embryo plus surrounding extraembryonic membranes), embryos, and yolk sacs had much lower hemoglobin concentrations than those observed in untreated SWV units, embryos, and yolk sacs. Additionally, cadmium exposure significantly decreased C57BL/6J embryonic hemoglobin levels on gestation day 10 (PM) and increased C57BL/6J yolk sac hemoglobin levels on gestation days 10 (AM) and 10 (PM). No difference in hemoglobin concentration was observed between untreated and cadmium-treated SWV embryos or yolk sacs. We propose that cadmium induces forelimb ectrodactyly by creating an acidotic embryonic environment and that the primary site at which cadmium exerts its teratogenic effect might be the yolk sac.