The effect of retinoic acid on positional memory in the dorsoventral axis of regenerating axolotl limbs

Abstract

We investigated the effect of retinoic acid (RA) on pattern regulation in the dorsoventral (DV) axis of regenerating axolotl limbs. Half and double half dorsal and ventral zeugopodia (lower arms or legs) were amputated through their distal ends, and 4 days later the animals were injected intraperitoneally with 50 (large animals) or 100 (small animals) micrograms RA/g body wt. Half and double half dorsal and ventral zeugopodia of uninjected axolotls, and sham-operated zeugopodia of untreated and RA-treated limbs served as controls. Skeletal patterns and the DV muscle patterns of control and experimental regenerates were then analyzed. Sham-operated zeugopodia of uninjected animals regenerated normally. Sham-operated, RA-treated zeugopodia regenerated normally with proximodistal duplications. Sixty percent of uninjected control dorsal half zeugopodia, 80% of control ventral half zeugopodia, and 100% of control double dorsal and double ventral zeugopodia regenerated distally, but the regenerates did not reconstitute the muscle pattern of the missing half. Thirty-eight percent of RA-treated ventral half zeugopodia and 78% of RA-treated double ventral zeugopodia failed to regenerate distally. Of those cases that did regenerate distally, none regenerated the muscle pattern of the missing half. By contrast, 100% of RA-treated dorsal half zeugopodia regenerated distally and all completed the normal DV muscle pattern. Forty-one percent of RA-treated double dorsal zeugopodia failed to regenerate, but of the remainder that did regenerate, 50% completed the normal DV muscle pattern. These represented eight cases, six of which regenerated single limbs, and two of which regenerated twin limbs, each with a normal DV muscle pattern. We interpret these data to mean that RA ventralizes the positional memory of blastema cells in the DV axis.