Determination of axial polarity in the urodele limb regeneration blastema

The state of determination of the anterior-posterior, dorsal-ventral and proximal-distal axes of the undifferentiated limb regeneration blastema was evaluated by heterografting and autografting experiments in which these axes were reversed with respect to the limb stump. Species-specific size differences in skeletal elements were used as markers to trace the origin of regenerate tissues in the heterografting experiments, and differences in the skeletal patterns of hindlimbs and forelimbs were used as markers in the autografting experiments. The resulting primary regenerates fell into two categories, those composed wholly or partly of donor tissues, and those composed entirely of host tissues. Regenerate structures formed from donor tissues always maintained the handedness of origin, while regenerates formed from host tissues always displayed host-side handedness. These results demonstrate that the axes of the blastema are determined from the start of regeneration, and that previous claims of axial lability in reversal experiments are based on an illusion created by resorption of graft tissue, accompanied by regeneration from the host. Reversal of the transverse axes resulted in the formation of supernumerary limbs. Analysis of heterograft cases in which the anterior-posterior axis was reversed showed that 50% of the supernumeraries were constructed partly of donor blastema tissue whose axial polarity was reversed with respect to the adjacent primary regenerate. The vast majority of the primary regenerates in these cases possessed the normal number of digits. It is thus likely that the reprogrammed donor blastema cells used to construct the supernumeraries are derived by division of a thin band of cells at the edge of the graft adjacent to the supernumerary.