Retinoid-induced limb defects 1: inhibition of cell proliferation in distal mesenchyme of limb buds in rats

Abstract

The present study was undertaken to investigate the effects of all- trans -retinoic acid (RA) on cell death and limb bud growth in forelimb buds and also to examine whether these events are involved in limb bone defects induced by RA in rats. RA was given at doses of 50 and 100 mg/kg to pregnant rats on Day 12 of pregnancy. Although RA did not show teratogenecity in the 50 mg/kg group, micromelia was observed in the 100 mg/kg group in all live fetuses on Day 21 of gestation. Micromelia was characterized by high incidences of proximodistal reduction of forearm bones without reduction of the humerus. The incidence of cell death in prechondrogenic areas, which differentiate into humerus and forearm bone, significantly increased 24 h after RA treatment in not only the 100 mg/kg, but also the 50 mg/kg, group. There was no difference in the incidence of cell death in the prechondrogenic area between the two groups. These observations indicate that the bone-specific defects were not the result of cell death alone in the prechondrogenic area. We examined the effects of RA on early forelimb bud growth, which is indispensable for the morphogenesis of the forelimb. Proximodistal length and protein content were decreased significantly in the forelimb bud 24 h after RA treatment at a dose of 100 mg/kg, but not 50 mg/kg. The immunohistochemical detection of bromodeoxyuridine (BrdU) incorporated into cells showed that at a dose of 100 mg/kg, cell proliferation was reduced in the distal mesenchyme, but not in the forearm-bone prechondrocytes of the forelimb bud. As the distal margin provides the cells differentiating into the prechondrocytes of future bones in the limb bud, these observations suggested that RA-induced inhibition of cell proliferation in the distal margin resulted in a decrease of forearm-bone prechondrocytes localized at more distal sites. We conclude that RA may inhibit the chondrogenesis of forearm bones by reducing cell proliferation in the distal margin of the forelimb bud, not by increasing cell death, and that this results in reduction defects in forearm bones.