Locomotory behavior of limb bud cells: Effect of excess vitamin A in vivo and in vitro

Limb buds excised from 11th to 12th day mouse embryos were cut at the junction of the shaft and autopod to produce proximal and distal segments respectively. The ectodermal covering of each segment was surgically removed and the core of mesenchymal tissue was diced into small fragments. Proximal and distal fragments were cultured on separate Falcon plastic flasks in a fluid medium consisting of Hams F 12 and fetal calf serum. The fragments attached and began to spread on the surface of the flask within 12 h after seeding. The morphology and rate of movement of the cells from the fragment were observed at 12, 36, and 60 h in culture. The cells of control proximal fragments were bipolar in outline for most of the 3-day culture period, while the cells obtained from distal segments changed from bipolar to stellate by the 2nd day of culture. The bipolar outline seemed to be a reliable indicator of cells that actively move in culture. The rates of migration were calculated and showed that the bipolar proximal cells were more motile than cells from distal limb areas. When previously untreated cells were cultured in medium containing 10 IU vitamin A acetate/ml both shape and motility were affected. The cells became stellate in outline by the end of the 1st day in culture. The rate of movement of the treated cells of both proximal and distal culture fragments was much below control levels. When embryos were treated in utero on day 11 1 2 with vitamin A acid (retinoic acid) and limb fragments prepared and cultured as described above, the cells exhibited a stellate shape and a decrease in migration tendencies during the first 12 h after seeding. By the end of the 3-day culture period the apparent recovery from retinoic acid treatment was demonstrated in both the morphology and the increased rate of movement of the cells. These results indicate that a difference between proximal and distal control cell migratory rates exists which, if manifest in the developing mouse limb bud, may play a role in the attainment of proper limb shape. Vitamin A seems capable of impairing those properties of cells necessary for cell movement which might be a factor involved in the production of the limb defects observed after maternal hypervitaminosis A in the mouse.