Morphological and molecular characterization of retinoic acid-induced limb duplications in mice

This study reports a morphological, skeletal, and molecular characterization of the supernumerary limbs induced by systemic administration of all-trans retinoic acid to egg-cylinder stage mouse embryos. As initially described by Rutledge et al. (Proc. Natl. Acad. Sci. USA 91, 5436, 1994), we have found that oral administration of all-trans retinoic acid (70 mg/kg body weight) at 5.5 days postcoitum induced the formation of supernumerary limbs. Most often, these arose as a pair of extra buds located caudally and ventrally to the normal (orthotopic) hindlimb buds without duplication of the lower body axis. The resulting one or two supernumerary hindlimbs were connected to an imperfectly mirror-image-duplicated pelvic girdle. Variable truncations of the stylopodium and zeugopodium skeleton, as well as abnormal splitting of the distal skeleton, were frequently observed. The apical ectodermal ridge of the extra limb buds expressed expected growth factor genes. However, an ectopic anterior expression of Sonic hedgehog and Hoxd-13 was seen in the supernumerary buds, suggesting that these buds would incorporate potential polarizing cells of the hindlimb or genital field and generate an ectopic polarizing zone. This is consistent with the reverse orientation of most supernumerary limbs at later stages. Some of the buds did not express limb-specific markers and were thus expected to degenerate or form nonlimb structures, as observed in an adult specimen. Less frequently, extra limb buds with normal polarity were associated to a duplicated lower body axis. Retinoic acid also generated a novel type of duplication in which "twin" hindlimbs with two parallel apical ectodermal ridges and zones of polarizing activity arose on one side of the embryo.