EFFECTS OF ISOTRETINOIN (13-CIS-RETINOIC ACID) ON THE DEVELOPMENT OF MOUSE LIMBS INVIVO AND INVITRO

Isotretinoin (13-cis-RA) is known to be teratogenic in humans and laboratory animals. The relatively low potency of 13-cis-RA in NRMI mice in comparison to the all-trans isomer has been proposed to be due to minimal transfer across the placenta (Creech-Kraft et al., '87). To further delineate the teratogenic potential of 13-cis-RA, a dose-response, temporal study was conducted in vivo and in vitro using submerged limb culture and image analysis evaluation of development. Dose-dependent embryotoxicity was produced by treatment on GD 7, while later treatments produced inconsistent effects on resorption rate and fetal weight. Treatment on either GD 7 or GD 8 produced a number of malformations in dose-dependent manner. Most common were tail and cleft palate defects, which were produced by 13-cis-RA on each of the days tested (GD 7-GD 11), with peak malformations occurring on GD 9 and GD 10 for tail and cleft palate, respectively. Most limb defects were produced after GD 10 and GD 11 exposure. The observed frequency of defects confirmed that in ICR mice 13-cis-RA is about 10-fold less potent than all-trans-RA as a limb teratogen (Kwasigroch and Kochhar, '80; Kochhar and Penner, '87). Effects observed via image analysis following maintenance of limbs in serum-free culture medium were dose dependent. Low dose treatment produced occasional polydactyly. The intermediate dose caused somewhat variable region-dependent increases in cartilaginous bone anlagen area. The high dose of 13-cis-RA produced irregular limb outlines, a reduction in bone anlagen area, and an inhibition of alcian blue staining of cartilage without affecting morphogenesis of bone anlagen. These results confirm that, when the effects of the administered doses are evaluated, 13-cis-RA is a much less potent teratogen in comparison to the all-trans isomer. More importantly, the results show that retinoids can enhance (at low and intermediate doses), depress (at high doses), or eliminate (high dose) chondrogenenic expression during limb morphogenesis in vitro. This indicates that retinoids such as 13-cis-RA can manipulate events in development in a variety of ways (i.e., produce malformations, interfere with chondrogenic expression without affecting morphogenesis, and stimulate growth) in a dose- and time-dependent manner. Although the ability of RA to act as a true morphogen has recently been questioned (Wanek et al., '91; Noji et al., '91),the results presented here support the position that RA can modulate the development of the limb (and probably other organ systems) in several vertebrate species.