Enhanced expression of limb malformations and axial skeleton alterations in legless mutants by transplacental exposure to retinoic acid.

This manuscript reports on the limb malformations and axial skeleton alterations found in legless fetuses and their heterozygote and wild-type littermates transplacentally exposed to all-trans-retinoic acid via a single intraperitoneal injection on Day 7, 8, 9, 9.5, 10, 10.5, or 11 of gestation. The most surprising aspect of the results was the temporal sensitivity of the legless mouse limb to exogenous retinoic acid. On Day 11, when both fore- and hindlimbs of nonmutant embryos can be made abnormal by retinoic acid and other teratogens, retinoic acid did not increase the frequency or severity of legless hindlimb defects and forelimb malformations were only slightly enhanced. On the other hand, retinoic acid administration on Day 7 exacerbated forelimb malformations in legless fetuses at a time when visible emergence of the affected structure is still 48 hr away. Heterozygote and wild-type fetuses had no limb malformations at this time point. A similar phenomenon was observed with hindlimb malformations after retinoic acid exposure on Day 8 except for a few mild limb malformations in heterozygotes at a high dose of retinoic acid. This early hypersensitivity of fore- and hindlimbs was followed by a period of reduced sensitivity (Day 8 forelimb; Day 9 hindlimb) when even very high doses (50 mg/kg) induced minimal changes in the typical legless malformation pattern. Subsequently, at the time of visible limb bud emergence (Day 9 forelimbs; Day 10 hindlimbs), sensitivity to exogenous retinoic acid was again detected. Surprisingly, the altered malformation patterns induced by retinoic acid in lgl mutants were nearly identical to those from earlier, pre-emergence exposure. A number of axial skeleton alterations were induced in legless fetuses by retinoic acid, especially after exposure on Days 7, 8, or 9. Posterior truncations were particularly noteworthy, showing a graded response in which frequency and severity of truncation were worst in lgl/lgl fetuses; heterozygotes gave an intermediate response, and wild-type fetuses were least affected. This exacerbation of the legless phenotype by exogenous retinoic acid coupled with the similarity between legless and retinoid malformations suggest that the legless mutation has altered endogenous retinoid homeostasis or a downstream retinoid-responsive gene.