Analysis of hoxa11 and hoxa13 expression during patternless limb regeneration in Xenopus

During limb regeneration, anuran tadpoles and urodele amphibians generate pattern-organizing, multipotent, mesenchymal blastema cells, which give rise to a replica of the lost limb including patterning in three dimensions. To facilitate the regeneration of nonregenerative limbs in other vertebrates, it is important to elucidate the molecular differences between blastema cells that can regenerate the pattern of limbs and those that cannot. In Xenopus froglet (soon after metamorphosis), an amputated limb generates blastema cells that do not produce proper patterning, resulting in a patternless regenerate, a spike, regardless of the amputation level. We found that re-expression of hoxa11 and hoxa13 in the froglet blastema is initiated although the subsequent proximal-distal patterning, including separation of the hoxa11 and hoxa13 expression domains, is disrupted. We also observed an absence of EphA4 gene expression in the froglet blastema and a failure of position-dependent cell sorting, which correlated with the altered hoxa11 and hoxa13 expression. Quantitative analysis of hoxa11 and hoxa13 expression revealed that hoxa13 transcript levels were reduced in the froglet blastema compared with the tadpole blastema. Moreover, the expression of sox9, an important regulator of chondrogenic differentiation, was detected earlier in patternless blastemas than in tadpole blastemas. These results suggest that appropriate spatial, temporal, and quantitative gene expression is necessary for pattern regeneration by blastema cells.