Meeting Abstract
Retinoic acid (RA), a vitamin A derivative, is involved in patterning during vertebrate limb development and urodele limb regeneration. Treatment with RA during limb regeneration can proximalize, posteriorize, and ventralize blastema cell positional memory and regenerate outcome. The purpose of this study was to take advantage of the green fluorescent protein (GFP) axolotl to visualize the regional stump contributions to regenerated limbs with and without RA treatment. To accomplish this, we grafted GFP anterior or posterior zeugopodial level half limbs to white sibling hosts to create two different limb morphologies: 1) sham-like normal limbs with either anterior or posterior half GFP allografts to white hosts; or 2) half GFP half white double anterior or double posterior limbs. As controls, anterior and posterior half zeugopod limbs were surgically created. Post healing time, all limbs were amputated at the wrist level, to visualize the cellular contributions of the GFP/white limb domains during regeneration. Four days post amputation, half the animals from each group were injected with either RA (150µg/gram body weight) to induce complete proximalization of the regenerate limb pattern, or 3µl/gram body weight dimethyl sulfoxide, the carrier solvent. Limbs were monitored and photographed periodically to determine the regional contributions of both anterior and posterior limb elements to the control and RA treated limbs. After regeneration, limbs were fixed, stained, and cleared to observe regenerate limb pattern. Our results support previous observations that the anterior half limb is not only responsive to RA, but also the major contributor to RA treated proximalized regenerate limbs.
